Methods for detecting b. anthracis infection

ABSTRACT

This invention pertains to methods for detecting  B. anthracis  and antibodies to  B. anthracis , the causative agent of anthrax, in a subject

FIELD OF THE INVENTION

This invention pertains to methods for detecting B. anthracis andantibodies to B. anthracis, the causative agent of anthrax, in asubject.

BACKGROUND OF THE INVENTION

Anthrax spores were first produced as weapons in the 1950s. Severalcountries including the former Soviet Union, the United States and Iraqare known to have produced anthrax weapons. Anthrax is a particularlyfearsome biological warfare agent, not only because of its deadliness,but also because anthrax weapons are relatively easy to produce anddeliver. Production of anthrax spores requires little more than basiclaboratory equipment and growth media. Anthrax weapons may be comprisedof an anthrax source and an industrial sprayer that can produce aerosolparticles of the appropriate size for victims to inhale. Such sprayers,for instance, can be mounted on low flying airplanes or other vehiclesand used to spread anthrax over a wide area. Because of the ease andrelatively small expense involved in producing and delivering anthraxweapons, such weapons are potentially highly attractive weapons of massdestruction for terrorist groups. Thus, in addition to potentialorganized military conflicts that may give rise to the use of suchweapons, terrorist organizations are a potential threat for the use ofsuch weapons in airports, office buildings and other centers of humanactivity.

Anthrax is caused by B. anthracis, a gram-positive, sporulatingbacillus. B. anthracis is a soil bacterium and is distributed worldwide.The organism exists in the infected host as a vegetative bacillus and inthe environment as a spore. The anthrax spore is typically the infectiveform of the bacterial life cycle. Anthrax spores can survive adverseenvironmental conditions and can remain viable for decades. Animals suchas cattle, sheep, goats and horses can contract the spores whilegrazing. Humans can contract anthrax from inoculation of minor skinlesions with spores from infected animals, their hides, wool or otherproducts, such as infected meat (Franz et al. (1997) J. Am. Med. Assoc.278(5): 399-411).

The typical mode of entry of the anthrax spore into the body,inhalation, results in an illness known as woolsorter's disease. Afterdeposit in the lower respiratory tract, spores are phagocytized bytissue macrophages and transported to hilar and mediastinal lymph nodes.The spores germinate into vegetative bacilli, producing a necrotizinghemorrhagic mediastinitis (Franz et al., supra). Symptoms include fever,malaise and fatigue, which can easily be confused with the flu. Thedisease may progress to an abrupt onset of severe respiratory distresswith dyspnea, stridor, diaphoresis and cyanosis. Death usually followswithin 24 to 36 hours.

Cutaneous anthrax infection occurs following external contact withanthrax. In cutaneous anthrax infection, symptoms include a skininfection that is distinguished by a raised itchy bump that resembles aninsect bite in the first days and develops into a vesicle and then ulcerwith a characteristic black necrotic area in the center. 20% ofuntreated cases of cutaneous infection result in death.

Gastrointestinal anthrax infection follows consumption of contaminatedmeat. Symptoms include nausea, loss of appetite, vomiting, fever,abdominal pain, vomiting of blood and severe diarrhea. Death results in25%-60% of cases.

Because the effects of exposure to anthrax are not immediate, andbecause the initial symptoms are easily confused with the flu, there isa need for a fast method to detect B. anthracis in a subject. This needis enhanced by the increasing number of anthrax threats that are calledinto governmental authorities each year and the recent transport ofanthrax through the United States Postal System. A fast method fordetermining whether a subject has been infected with anthrax is,therefore, essential.

Anthrax spores have S-layers, as do spores of many other archea andbacteria. Most S-layers are comprised of repeats of a single protein(Etienne-Toumelin et al., J. Bacteriol. 177:614-20 (1995)). The S-layerof B. anthracis, however, is comprised of at least two proteins: EA1(Mesnage et al., Molec. Microbiol. 23:1147-55 (1997)) and surface arrayprotein (SAP) (see Etienne-Toumelin, et al., supra). Fully virulent B.anthracis isolates are encapsulated by a capsule that encompasses theS-layer of the bacteria and prevents access of antibodies to both EA1and SAP (Mesnage et al., J. Bacteriol. 180:52-58 (1998)). Amino acids180 to 700 of SEQ ID NO:1 are specific for SAP.

A fast and efficient method is needed to detect infection of animals,and especially humans, by anthrax. The present invention addresses thisand other problems.

SUMMARY OF THE INVENTION

The present invention provides novel methods of detecting antibodies andantigens to B. anthracis. In one embodiment of the invention, a methodfor the detection of an anti-B. anthracis antibody present in abiological sample comprises two steps. The first step comprisescontacting a biological sample from an animal with an affinity agentcomprising an epitope recognized by an antibody that specifically bindsto SEQ ID NO:1, wherein the affinity agent forms a complex with theanti-B. anthracis antibody if the anti-B. anthracis antibody is presentin the sample. The second step comprises detecting the presence orabsence of the complex, wherein the presence of the complex indicatesthe presence of antibodies to B. anthracis in the sample.

In one embodiment of the invention, the complex is detected prior toclinical manifestation of anthrax in the animal.

In one embodiment of the invention, the affinity agent comprises apolypeptide at least 80% identical to SEQ ID NO:1 or a fragment of SEQID NO:1 at least 10 amino acids long. In another embodiment, theaffinity agent comprises SEQ ID NO:1. In yet another embodiment, theaffinity agent comprises a polypeptide at least 80% identical to aminoacids 180 to 700 of SEQ ID NO:1 or a fragment of amino acids 180 to 700of SEQ ID NO:1 at least 10 amino acids long.

In one aspect of the invention, the animal contacted is a human. Inanother aspect of the invention, the biological sample taken from theanimal is bodily fluid. In one aspect of the invention, the bodily fluidis blood which may be further processed to serum or plasma.

In one embodiment of the invention, the affinity agent is immobilized ona solid support. In one aspect of the invention, the solid support is amicrotiter dish. In another embodiment, the method for the detection ofan anti B. anthracis antibody further comprises contacting the complexwith an antibody that binds to the complex. In one aspect of theinvention, the antibody is labeled. In another aspect, the label isselected from the group consisting of enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds and bioluminescent compounds. In yet anotheraspect of the invention, the antibody specifically binds to a humanantibody.

In one embodiment of the invention, the anti-B. anthracis antibodydetected is an IgG isotype. In another embodiment, the anti-B. anthracisantibody detected is an IgM isotype. In yet another embodiment of theinvention, the anti-B. anthracis antibodies detected comprise IgG andIgM isotypes.

In one embodiment of the invention, the method further comprises thesteps of contacting the biological sample with a capture reagent,wherein the capture reagent forms a complex with a B. anthracis surfacearray protein if the surface array protein is present in the sample, anddetecting the presence or absence of the complex.

In one aspect of the invention the biological sample is blood or plasma.

In one embodiment of the invention, the surface array protein comprisesa polypeptide having an amino acid sequence at least 80% identical toSEQ ID NO:1 or a fragment of SEQ ID NO:1 at least 10 amino acids long.In another embodiment, the polypeptide comprises SEQ ID NO:1. In yetanother embodiment, the surface array protein comprises a polypeptidehaving an amino acid sequence at least 80% identical to amino acids 180to 700 of SEQ ID NO:1 or a fragment of amino acids 180 to 700 of SEQ IDNO:1 at least 10 amino acids long

In one embodiment of the invention, the capture reagent comprises anantibody that binds to SEQ ID NO:1. In one aspect of the invention, thecapture reagent is a recombinant antibody. In another aspect, thecapture reagent is a recombinant polyclonal antibody. In yet anotheraspect, the capture reagent is a monoclonal antibody.

In one embodiment, the capture reagent is immobilized on a solidsupport. In one embodiment of the invention, the capture reagent isimmobilized on the same solid support as the affinity agent. In anotherembodiment, the solid support is a microtiter dish.

The present invention further provides a method for detecting thesurface array protein comprising contacting the surface array proteinwith a detection reagent that can bind to the surface array protein. Inone aspect of the invention, the detection reagent is an antibody thatbinds to the complex. In one embodiment, the detection reagent islabeled.

The invention also provides a kit for the detection of an anti-B.anthracis antibody in a biological sample. In some embodiments, the kitcomprises an affinity agent immobilized on a solid support. In someembodiments, the affinity agent comprises an epitope recognized by anantibody that specifically binds to SEQ ID NO:1, wherein the affinityagent forms a complex with the anti-B. anthracis antibody if the anti-B.anthracis antibody is contacted to the affinity agent. In one embodimentof the invention, the affinity agent comprises SEQ ID NO:1 or a fragmentof SEQ ID NO:1 at least 10 amino acids long. In yet another embodiment,the affinity agent comprises a polypeptide at least 80% identical toamino acids 180 to 700 of SEQ ID NO:1 or a fragment of amino acids 180to 700 of SEQ ID NO:1 at least 10 amino acids long.

In one aspect of the invention, the solid support provided in the kitcomprises a microtiter plate and the affinity agent is present in thewells of the microtiter plate.

In one embodiment of the invention, the kit comprises a detectionreagent. In one aspect, the detection reagent provided in the kitcomprises an antibody that binds to the complex. In another aspect, theantibody is labeled. In yet another aspect, the label is selected fromthe group consisting of enzymes, radioisotopes, fluorescent compounds,colloidal metals, chemiluminescent compounds, phosphorescent compoundsand bioluminescent compounds.

In one embodiment of the invention, the antibody provided in the kitspecifically binds to a human antibody. In one aspect of the invention,the human antibody detected comprises IgG and IgM isotypes.

In another embodiment of the invention, the kit further comprises acapture reagent immobilized on a solid support, wherein the capturereagent forms a complex with a B. anthracis surface array protein if thesurface array protein is present in the sample. In one aspect, thecapture reagent and affinity agent are immobilized on the same solidsupport. In another aspect, the capture reagent is immobilized on amicrotiter dish.

In one embodiment of the invention, the capture reagent provided in thekit is an antibody. In one aspect, the capture reagent is a recombinantpolyclonal antibody. In yet another aspect, the capture reagent is amonoclonal antibody.

In another embodiment, the kit further comprises a positive control thatcomprises a polypeptide that comprises an antigenic determinant of a B.anthracis surface array protein. In one aspect of the invention, theantigenic determinant comprises an amino acid sequence of SEQ ID NO:1 ora fragment of SEQ ID NO:1 at least 10 amino acids long.

In yet another aspect, the antigenic determinant comprises a polypeptideat least 80% identical to amino acids 180 to 700 of SEQ ID NO:1 or afragment of amino acids 180 to 700 of SEQ ID NO:1 at least 10 aminoacids long.

In one embodiment of the invention, the kit comprises a detectionreagent. In one aspect, the detection reagent comprises an antibody thatbinds to the complex. In another aspect of the invention, the antibodyis labeled.

DETAILED DESCRIPTION I. Introduction

The present invention provides methods for detecting B. anthracisinfection in an animal. More specifically, this invention providesmethods for detecting antibodies that specifically bind to a B.anthracis surface array protein (SAP) in an animal. B. anthracis SAP isan antigen or antigenic determinant that is specific for B. anthracis.Therefore, detection of antibodies in a biological sample thatspecifically bind to SAP indicate that an animal has been exposed to B.anthracis and may be infected with anthrax.

The present invention also provides methods for detecting the B.anthracis surface array protein in an animal. The B. anthracis SAPpolypeptide in the animal can be an antigen or antigenic determinantthat is specific for B. anthracis. Detecting B. anthracis surface arrayprotein in an animal also indicates that an animal has been exposed toB. anthracis and may be infected with anthrax.

II. Definitions

The phrase “capture reagent” refers to a molecule that specificallybinds to a surface array protein of B. anthracis or a portion thereof.Capture reagents include naturally and non-naturally-occurring moleculesthat can specifically bind a target molecule. For instance, antibodies,as well as peptides that specifically bind a target molecule and aredeveloped using phage display or other combinatorial system areencompassed by this definition.

The phrase “affinity agent” refers to a molecule that specifically bindsto antibodies specific for a B. anthracis surface array protein.Affinity agents include naturally- and non-naturally-occurring moleculesthat can specifically bind to such antibodies. Affinity agents, include,any type of molecule recognized by antibodies specific for SAP,including, e.g., polypeptides.

The phrase “surface array polypeptide” or “SAP polypeptide” refers to apolypeptide associated with the S-layer of B. anthracis. SAPpolypeptides are typically one of the most abundant, endogenouspolypeptides in B. anthracis. See, e.g., Etienne-Toumelin et al., J.Bacteriol. 177:614-620 (1995). Exemplary SAP polypeptides include, e.g.,SEQ ID NO:1.

A “biological sample” as used herein is a sample of biological tissue orfluid that contains an analyte (such as, antibodies or antigens ofinterest). These samples can be tested by the methods described hereinand include human and animal body fluids such as whole blood, serum,plasma, cerebrospinal fluid, urine, lymph fluids, and various externalsecretions of the respiratory, intestinal and genitourinary tracts,tears, saliva, milk, white blood cells, myelomas, and the like; andbiological fluids such as cell culture supernatants; fixed tissuespecimens; and fixed cell specimens. Biological samples may also includesections of tissues such as biopsy and autopsy samples or frozensections taken for histologic purposes. A biological sample is typicallyobtained from a eukaryotic organism, most preferably a mammal such as aprimate, e.g., human or chimpanzee; cow; dog; cat; a rodent, e.g.,guinea pig, rat, mouse; rabbit; or other mammal; or a bird; reptile; orfish. A biological sample can be from a laboratory source or from anon-laboratory source that is either known or not known to contain B.anthracis or antibodies to B. anthracis.

The term “analyte” refers to the substance to be detected that may bepresent in the sample. The analyte can be any substance for which thereexists a naturally occurring specific binding member (such as anantibody or antigen), or for which a specific binding member can beprepared. Thus, an analyte is a substance that can bind to one or morespecific binding members in an assay. “Analyte” also includes anyantigenic substances, haptens, antibodies, and combinations thereof. Theanalyte can include a protein, a peptide, an amino acid, a nucleotidetarget, and the like.

The phrases “specifically binds to” or “specifically immunoreactivewith”, when referring to an antibody or other binding moiety, such asthe capture reagents or affinity agents described herein, refers to abinding reaction which is determinative of the presence of a targetanalyte in the presence of a heterogeneous population of proteins andother biologics. Thus, under designated assay conditions, the specifiedbinding moieties, e.g., capture reagents or affinity agents, bindpreferentially to a particular target analyte and do not bind in asignificant amount to other components present in a sample. Specificbinding to a target analyte under such conditions may require a bindingmoiety that is selected for its specificity for a particular targetanalyte. Such a binding moiety might include a particular epitopespecifically immunoreactive with a particular antibody. Typically aspecific or selective reaction will be at least twice background signalor noise and more typically more than 10 to 100 times background.

The term “epitope” means an antigenic determinant that is capable ofspecific binding to an antibody. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents. Epitopes can include non-contiguous amino acids, aswell as contiguous amino acids.

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen or antibody). Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition,Raven Press, NY (1993)). While various antibody fragments are defined interms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv).

The terms “peptidomimetic” and “mimetic” refer to a synthetic chemicalcompound that has substantially the same structural and functionalcharacteristics of a polypeptide, e.g., a SAP polypeptide. Peptideanalogs are commonly used in the pharmaceutical industry as non-peptidedrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber andFreidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem. 30:1229(1987), which are incorporated herein by reference). Peptide mimeticsthat are structurally similar to therapeutically useful peptides may beused to produce an equivalent or enhanced therapeutic or prophylacticeffect. Generally, peptidomimetics are structurally similar to aparadigm polypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as SAP polypeptide, but have one or morepeptide linkages optionally replaced by a linkage selected from thegroup consisting of, e.g., —CH2NH—, —CH2S—, —CH2-CH2-, —CH═CH— (cis andtrans), —COCH2-, —CH(OH)CH2-, and —CH2SO—. The mimetic can be eitherentirely composed of synthetic, non-natural analogues of amino acids,or, is a chimeric molecule of partly natural peptide amino acids andpartly non-natural analogs of amino acids. The mimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or activity. For example, a mimetic compositionis within the scope of the invention if it is capable of carrying outthe binding or antigenic activities of SAP.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames that flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

The term “nucleic acid” or “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions) andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka etal., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992);Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleicacid is used interchangeably with gene, cDNA, and mRNA encoded by agene.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull-length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either the commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein that encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecomplement of a test sequence. Optionally, the identity exists over aregion that is at least about 50 nucleotides in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotidesin length.

The term “similarity,” or percent “similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined in the 8 conservativeamino acid substitutions defined above (i.e., 60%, optionally 65%, 70%,75%, 80%, 85%, 90%, or 95% similar over a specified region), whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Such sequences are then said to be “substantially similar.”Optionally, this identity exists over a region that is at least about 50amino acids in length, or more preferably over a region that is at leastabout 100 to 500 or 1000 or more amino acids in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Ausubelet al., Current Protocols in Molecular Biology (1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal. (1984) Nuc. Acids Res. 12:387-395).

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42°C., or 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and0.1% SDS at 65° C. Such washes can be performed for 5, 15, 30, 60, 120,or more minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Such washes can be performed for 5, 15,30, 60, 120, or more minutes. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

The phrase “a nucleic acid sequence encoding” refers to a nucleic acidwhich contains sequence information for a structural RNA such as rRNA, atRNA, or the primary amino acid sequence of a specific protein orpeptide, or a binding site for a trans-acting regulatory agent. Thisphrase specifically encompasses degenerate codons (i.e., differentcodons which encode a single amino acid) of the native sequence orsequences which may be introduced to conform with codon preference in aspecific host cell.

The term “reactive” means capable of binding or otherwise associatingnonrandomly with a binding pair.

The term “label” refers to a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include radioactive reagents, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in ELISA),biotin, dioxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available.

A “labeled antibody” is one that is bound, either covalently, through alinker, or through ionic, van der Waals or hydrogen bonds to a labelsuch that the presence of the antibody may be detected by detecting thepresence of the label bound to the antibody.

The phrase “complex” as used herein, refers to any distinct chemicalspecies in which two or more identical or nonidentical chemical species(ionic or uncharged) are associated.

III. Affinity Agents of the Invention

The present invention provides affinity agents that are capable ofspecifically binding antibodies specific for SAP. The methods of thepresent invention employ affinity agents containing one or more SAPepitopes as binding reagents that specifically bind to antibodies to B.anthracis. Affinity agents can be, e.g., polypeptides, peptidomimeticcompounds, or other molecules such as haptens that can be bound by ananti-SAP antibody. Polypeptides such as SAP polypeptides and polypeptidefragments thereof that contain at least one epitope specific for SAP areuseful affinity agents. Polypeptides other than SAP that contain one ormore SAP epitopes are useful affinity agents as well.

1. SAP Polypeptides

Peptides containing antigenic determinants of SAP can be produced bymethods known to those of skill in the art. The amino acid sequence of aB. anthracis SAP polypeptide is provided as SEQ ID NO:1. A B. anthracisSAP polypeptide from a different strain is described in Etienne-Toumelinet al., J. Bacteriol. 177:614-620 (1995).

The SAP proteins, or subsequences thereof, may be synthesized usingrecombinant DNA methodology. Generally this involves creating a DNAsequence that encodes the polypeptide, modified as desired, placing theDNA in an expression cassette under the control of a particularpromoter, expressing the protein in a host, isolating the expressedprotein and, if required, renaturing the protein. Alternatively,endogenous SAP polypeptides can be isolated from B. anthracis.

SAP polypeptides can be expressed in a variety of host cells, includingE. coli, other bacterial hosts, yeasts, filamentous fungi, and varioushigher eukaryotic cells such as the COS, CHO and HeLa cells lines andmyeloma cell lines. Techniques for gene expression in microorganisms aredescribed in, for example, Smith, Gene Expression in RecombinantMicroorganisms (Bioprocess Technology, Vol. 22), Marcel Dekker, 1994.Examples of bacteria that are useful for expression include, but are notlimited to, Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus,Pseudomonas, Klebsielia, Proteus, Salmonella, Serratia, Shigella,Rhizobia, Vitreoscilla, and Paracoccus. Filamentous fungi that areuseful as expression hosts include, for example, the following genera:Aspergillus, Trichoderma, Neurospora, Penicillium, Cephalosporium,Achlya, Podospora, Mucor, Cochliobolus, and Pyricularia. See, e.g., U.S.Pat. No. 5,679,543 and Stahl and Tudzynski, Eds., Molecular Biology inFilamentous Fungi, John Wiley & Sons, 1992. Synthesis of heterologousproteins in yeast is well known and described in the literature. Methodsin Yeast Genetics, Sherman, F., et al., Cold Spring Harbor Laboratory,(1982) is a well recognized work describing the various methodsavailable to produce the enzymes in yeast.

SAP proteins, whether recombinantly or naturally produced, can bepurified, either partially or substantially to homogeneity, according tostandard procedures of the art, such as, for example, ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes, ProteinPurification, Springer-Verlag, N.Y. (1982), Deutscher, Methods inEnzymology Vol. 182: Guide to Protein Purfication., Academic Press, Inc.N.Y. (1990)). Once purified, partially or to homogeneity as desired, thepolypeptides can then be used (e.g., as affinity agents or as immunogensfor antibody production).

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the SAP protein(s) may possess aconformation substantially different than the native conformations ofthe constituent polypeptides. In this case, it may be necessary ordesirable to denature and reduce the polypeptide and then to cause thepolypeptide to re-fold into the preferred conformation. Methods ofreducing and denaturing proteins and inducing re-folding are well knownto those of skill in the art (See, Debinski et al. (1993) J. Biol. Chem.268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem. 4:581-585; and Buchner et al. (1992) Anal. Biochem. 205: 263-270).Debinski et al., for example, describe the denaturation and reduction ofinclusion body proteins in guanidine-DTE. The protein is then refoldedin a redox buffer containing oxidized glutathione and L-arginine.

One of skill also would recognize that modifications can be made to theSAP polypeptides without diminishing their antigenic activity. The SAPpolypeptides need only contain one epitope specific for SAP.Polypeptides other than SAP that contain one or more SAP epitopes can beproduced in the same way. Modifications can be made to facilitate thecloning, expression, or incorporation of the polypeptide into a fusionprotein. Such modifications include, for example, a methionine added atthe amino terminus to provide an initiation site, or additional aminoacids (e.g., poly His) placed on either terminus to create convenientlylocated restriction sites or termination codons or purificationsequences.

2. B. anthracis SAP-Encoding Nucleic Acids.

Nucleic acids that encode B. anthracis are useful for the recombinantproduction of SAP. Such nucleic acids can be isolated, for example, byroutine cloning methods. The cDNA sequence provided in SEQ ID NO:2 canbe used to provide probes that specifically hybridize to a SAP gene, toa SAP mRNA, or to a SAP cDNA in a cDNA library (e.g., in a Southern orNorthern blot). Once the target SAP nucleic acid is identified, it canbe isolated according to standard methods (see, e.g., Sambrook, Berger,and Ausubel, supra.).

SAP nucleic acids also can be isolated by amplification methods such aspolymerase chain reaction (PCR), the ligase chain reaction (LCR), thetranscription-based amplification system (TAS), the self-sustainedsequence replication system (SSR) and other cloning and in vitroamplification methodologies. Examples of these techniques andinstructions sufficient to direct persons of skill through many cloningexercises are found in Berger, Sambrook, and Ausubel (all supra.);Cashion et al., U.S. Pat. No. 5,017,478; and Carr, European Patent No.0,246,864. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods are found in Berger, Sambrook,and Ausubel, as well as Mullis et al. (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al., eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3: 81-94;(Kwoh et al. (1989) Proc. Nat'l. Acad. Sci. USA 86: 1173; Guatelli etal. (1990) Proc. Natl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J.Clin. Chem. 35: 1826; Landegren et al. (1988) Science 241: 1077-1080;Van Brunt (1990) Biotechnology 8: 291-294; Wu and Wallace (1989) Gene,4: 560; and Barringer et al. (1990) Gene 89: 117.

A polynucleotide that encodes a polypeptide containing an antigenicdeterminant of SAP, e.g., a SAP polypeptide such as SEQ ID NO:1, can beoperably linked to appropriate expression control sequences for aparticular host cell in which the polypeptide is to be expressed. Suchconstructs are often referred to as “expression cassettes.” For E. coli,appropriate control sequences include a promoter such as the T7, trp, orlambda promoters, a ribosome binding site and preferably a transcriptiontermination signal. For eukaryotic cells, the control sequencestypically include a promoter which optionally includes an enhancerderived from immunoglobulin genes, SV40, cytomegalovirus, etc., and apolyadenylation sequence, and may include splice donor and acceptorsequences. In yeast, convenient promoters include GAL1,10 (Johnson andDavies (1984) Mol. Cell. Biol. 4:1440-1448) ADH2 (Russell et al. (1983)J. Biol. Chem. 258:2674-2682), PHO5 (EMBO J. (1982) 6:675-680), andMFocl (Herskowitz and Oshima (1982) in The Molecular Biology of theYeast Saccharomyces (eds. Strathem, Jones, and Broach) Cold SpringHarbor Lab., Cold Spring Harbor, N.Y., pp. 181-209).

Expression cassettes are typically introduced into a vector thatfacilitates entry into a host cell, and maintenance of the expressioncassette in the host cell. Vectors that include a polynucleotide thatencodes a polypeptide containing a SAP epitope are provided by theinvention. Such vectors often include an expression cassette that candrive expression of the SAP polypeptide. To easily obtain a vector ofthe invention, one can clone a polynucleotide that encodes the SAPpolypeptide into a commercially or commonly available vector. A varietyof common vectors suitable for this purpose are well known in the art.For cloning in bacteria, common vectors include pBR322 derived vectorssuch as PBLUESCRIPT™, and λ-phage derived vectors. In yeast, vectorsinclude Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicatingplasmids (the YRp series plasmids) and pGPD-2. A multicopy plasmid withselective markers such as Leu-2, URA-3, Trp-1, and His-3 is alsocommonly used. A number of yeast expression plasmids such as YEp6,YEp13, YEp4 can be used as expression vectors. The above-mentionedplasmids have been fully described in the literature (Botstein et al.(1979) Gene 8:17-24; Broach et al. (1979) Gene, 8:121-133). For adiscussion of yeast expression plasmids, see, e.g., Parents, B., YEAST(1985), and Ausubel, Sambrook, and Berger, all supra). Expression inmammalian cells can be achieved using a variety of commonly availableplasmids, including pSV2, pBC12BI, and p91023, as well as lytic virusvectors (e.g., vaccinia virus, adenovirus, and baculovirus), episomalvirus vectors (e.g., bovine papillomavirus), and retroviral vectors(e.g., murine retroviruses).

The nucleic acids that encode SAP polypeptides or other polypeptidescontaining SAP epitopes can be transferred into the chosen host cell bywell-known methods such as calcium chloride transformation for E. coliand calcium phosphate treatment or electroporation for E. coli ormammalian cells. Cells transformed by the plasmids can be selected byresistance to antibiotics conferred by genes contained on the plasmids,such as the amp, gpt, neo and hyg genes, among others. Techniques fortransforming fungi are well known in the literature and have beendescribed, for instance, by Beggs et al. ((1978) Proc. Natl. Acad. Sci.USA 75: 1929-1933), Yelton et al. ((1984) Proc. Natl. Acad. Sci. USA 81:1740-1747), and Russell ((1983) Nature 301: 167-169). Procedures fortransforming yeast are also well known (see, e.g., Beggs (1978) Nature(London), 275:104-109; and Hinnen et al. (1978) Proc. Natl. Acad. Sci.USA, 75:1929-1933. Transformation and infection methods for mammalianand other cells are described in Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology 152 Academic Press, Inc., SanDiego, Calif. (Berger); Sambrook et al. (1989) Molecular Cloning—ALaboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor Press, NY, (Sambrook et al.); Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (1994 Supplement) (Ausubel).

Once, peptides containing SAP epitopes are produced, they can be used asaffinity agents to detect antibodies present in a biological sample.

IV. Detecting Antibody with an Affinity Agent

In the present invention, affinity agents are used to detect antibodiesspecific for B. anthracis. After assaying for antibodies to B. anthracisin a sample with an affinity agent, a diagnosis of anthrax infection canbe made. If the affinity agent contacts antibodies specific for B.anthracis in a sample, a complex of affinity agent and antibodies willform. If antibodies specific for B. anthracis are not present in thesample, the affinity agent will not specifically bind to antibodies andno complex will form. The presence of the complex indicates exposure toB. anthracis.

The methods of the present invention employ different immunologictechniques and immunoassays, including Western blotting, to detectantibodies to B. anthracis in a sample. A general overview of theapplicable technology can be found in Harlow & Lane, Antibodies: ALaboratory Manual (1988).

One method of detection is the enzyme-linked immunosorbent assay knownas ELISA. In this method, antibodies in a sample are detected after theantibodies are specifically bound to an affinity agent on a solidsupport. A reagent (e.g., an affinity agent) that specifically binds tothe antibodies can be non-diffusively immobilized on the support eitherby covalent or non-covalent methods, which are known to those of skillin the art. See, e.g., Pluskal et al. (1986) Biotechniques 4:272-283.Suitable supports include, for example, glasses, plastics, polymers,metals, metalloids, ceramics, organics, and the like. Specific examples,include, but are not limited to, microtiter plates, nitrocellulosemembranes, nylon membranes, and derivatized nylon membranes, beads, andalso particles, such as agarose, SEPHADEX™, and the like. Assay systemsfor use in the methods and kits of the invention include, but are notlimited to, dipstick-type devices, immunochromatographic test strips andradial partition immunoassay devices, microtiter assays and flow-throughdevices. Where the solid support is a membrane, the test sample can flowthrough the membrane, for example, by gravity, capillary action, orunder positive or negative pressure.

Once the affinity agent is immobilized on the solid support, theimmobilized affinity agent is contacted with the sample suspected ofcontaining the antibody. After a suitable reaction time, unboundcomponents are removed by washing. An enzyme-conjugated secondaryanti-isotype antibody is then added which binds to humanimmunoglobulins. The enzyme is preferably, but not limited to, eitherhorseradish peroxidase, alkaline phosphatase, or beta-galactosidase. Theenzyme is capable of converting a colorless or near colorless substrateor co-substrate into a highly colored product or a product capable offorming a colored complex with a chromogen. Alternatively, the detectionsystem or assay may employ an enzyme which, in the presence of theproper substrate(s), emits light. The amount of product formed can bedetected either visually, spectrophotometrically, electrochemically, orluminometrically, and is compared to a similarly treated control. Thedetection system may also employ radioactively labeled antibodies, inwhich case the amount of immune complex is quantified by scintillationcounting or gamma counting.

Another method of detection is by competition assay. In competitionassays, a sample, such as sera, from the subject is reacted with anaffinity agent bound to a solid support which may be, for example, aplastic bead or tube or ELISA 96-well plate. Excess sera is washed away.A labeled (enzyme linked, fluorescent, radioactive, etc.) monoclonalantibody that binds to the affinity reagent is then contacted with thesolid support. The amount of inhibition of monoclonal antibody bindingis measured relative to a control in order to determine whetherantibodies are present in the sera.

By the same token, antibodies that bind the affinity agent may be boundto the solid support. Labeled affinity agent may be mixed with suitabledilutions of the sample to be tested. This mixture is then brought intocontact with the antibody bound to the solid support. After a suitableincubation period, the solid support is washed and the amount of labeledaffinity agent is quantified. A reduction in the amount of label boundto the solid support is indicative of the presence of antibodiesspecific for the affinity agent in the original sample.

Another method of detection is the homogenous immunoassay. With thisassay, there are many variations in design. By way of example, numerouspossible configurations for homogeneous enzyme immunoassays and methodsby which they may be performed are given in Tijssen, P., Practice andTheory of immunoassays, Elsevier Press, Amersham, Oxford, N.Y., 1985.Detection systems which may be employed include those based on enzymechanneling, bioluminescence, allosteric activation and allostericinhibition. Methods employing liposome-entrapped enzymes or coenzymesmay also be used (see e.g., Pinnaduwage, P. and Huang, L., Clin. Chem.(1988) 34/2: 268-272, and Ullmann, E. F. et al., Clin Chem. (1987) 33/9:1579-1584.)

Another method of detection is the micro-agglutination assay. In thisassay, latex beads, red blood cells or other agglutinable particles arecoated with the affinity agent and mixed with a sample from the subject,such that antibodies in the sample that are specifically reactive withthe affinity agent crosslink with the antigen, causing agglutination.The agglutinated affinity agent-antibody complexes form a precipitate,visible with the naked eye or by spectrophotometer.

Membrane-based detection methods may be used to detect antibodies to B.anthracis. These methods are described in, e.g., U.S. Pat. No.5,922,615. These systems employ an apparatus that includes a porousmember, such as a membrane or a filter, onto which is bound amultiplicity of affinity agents that specifically bind antibodies to B.anthracis. The apparatus also includes a non-absorbent member with atextured surface in communication with the lower surface of the porousmember. The textured surface of the non-absorbent member can be agrooved surface (e.g., analogous to the surface of a record album) or itcan be composed of channels, such that when the porous and non-absorbentmembers are brought into contact with one another a network of capillarychannels is formed. The capillary network is formed from the contact ofthe porous member with the textured surface of the non-absorbent memberand can be constructed either before or subsequent to the initialcontacting of the porous member with a fluid.

In some embodiments, the capillary communication between the porousmember and the non-absorbent member favors delaying the transferal offluid from the porous member to the capillary network formed by theporous member and the textured surface of the non-absorbent member untilthe volume of the added fluid substantially exceeds the void volume ofthe porous member. The transferal of fluid from the porous member to thenetwork of capillary channels formed by the porous member and thetextured surface of the non-absorbent member can occur without the useof external means, such as positive external pressure or vacuum, orcontact with an absorbent material.

An optional member which is placed in contact with the upper surface ofthe porous member may be used to partition the upper surface of thedevice into discrete openings. Such openings can access either theporous member or the textured surface of the non-absorbent secondmember. The optional member can in conjunction with the non-absorbentmember compose a fluid receiving zone in which there is no interveningporous member. A fluid receiving zone constructed from the non-absorbentmember and the optional member provides fluid capacity in addition tothat provided by the network of capillary channels created by thecontact of the porous member and the non-absorbent member. The openingsin the optional member may include a first fluid opening and also anadditional fluid opening. The first fluid opening functions as a portalfor the introduction of the first fluid added to the device. Theadditional fluid opening serves as an additional portal through whichadditional fluids may be added to the inventive device.

To perform an assay using these devices, a volume of the test sample isadded to the porous member, where the sample permeates the void volumeof the porous member and thereby contacts the affinity agent immobilizedon the porous member. In a non-competitive assay, the sample to beassayed is applied to the porous member and the antibodies, if present,are bound by the affinity agent. A detection reagent for the antibodiesis then added as an additional fluid; these bind to the complex of theantibodies and affinity agent. An additional fluid containing reagentsto effect a separation of free from bound labeled reagents can be addedto remove excess detection reagent, if needed.

This device is designed to provide sufficient sensitivity to measure lowconcentrations of antibodies specific for SAP or for an antigenicdeterminant of SAP because one can use large amounts of sample andefficiently remove the excess of detection reagent. Indeed, theefficient separation of free from bound label achieved by the network ofcapillary channels of this device improves the discrimination ofspecific SAP antibodies-associated signal over non-specific backgroundsignal. If needed, a signal developer solution is then added to enablethe label of the detection moiety to develop a detectable signal. Thesignal developed can then be related to the concentration of the targetligand within the sample. In one embodiment, the transfer of fluidbetween the porous first member of the device and the network ofcapillary channels formed by the contact of the porous member andtextured surface of the non-absorbent second member of the device isgenerally self-initiated at the point when the total volume of fluidadded to the device exceeds the void volume of the porous member, thusobviating the need for active interaction by the user to remove excessfluid from the analyte detection zone. This method enables the detectionof antibodies specific for SAP in a manner that is simple, rapid,convenient, sensitive and efficient in the use of reagents.

The labels used in the detection systems allow detection by a variety ofmethods including but not limited to visual detection of a precipitateor color change, visual detection by microscopy, automated detection byspectrometry, radiometric measurement, sorting by flow cytometry, or thelike. Examples of detectable labels include fluorescein and rhodamine(for fluorescence microscopy), horseradish peroxidase (for either lightor electron microscopy and biochemical detection), biotin-streptavidin(for light or electron microscopy) and alkaline phosphatase (forbiochemical detection by color change).

V. Antibody Response

An animal, e.g., a human, that is exposed to an antigenic determinant ofB. anthracis will, at some time following exposure, begin makingantibodies to B. anthracis. There are five classes of antibodies (IgM,IgG, IgD, IgE, IgA) that can be made in an immunogenic response to anantigen. All five classes of antibodies can be detected by the methodsand kits of this invention. IgM is the first class of antibody to appearon the surface of a developing B cell. It is the first antibody producedin response to antigenic determinants. IgM antibodies are not secretedin large quantities and generally have low affinity, yet they arecapable of efficiently binding antigen. In the early stages of a primaryantibody response, IgM is the only antibody secreted into the blood. IgMantibodies are, therefore, indicative of recent antigenic exposure.

One of the first serological markers of anthrax infection in an animalis IgM antibodies specific for B. anthracis. The present inventionprovides methods for detecting IgM antibodies before clinicalmanifestation of disease in the animal. The affinity agents of thisinvention, i.e., SAP polypeptides, are capable of efficiently bindingIgM antibodies present in a biological sample. In some animals, IgMantibodies may be the only class of antibodies present in the samplethat indicate anthrax infection. Determination of anti-B. anthracisantibodies of the IgM class to B. anthracis is, therefore, useful forearly detection of anthrax infection in an animal.

After producing IgM antibodies, antigen-stimulated B cells in an animalexposed to B. anthracis may produce IgD and IgG antibodies. Otherantigen-stimulated cells may produce IgG, IgE or IgA antibodies. IgG,IgE and IgA molecules are referred to as secondary classes ofantibodies. Another aspect of the present invention, therefore, is thedetection of complexes between an affinity agent and antibodies otherthan IgM. The presence of IgG or other classes of antibodies bound tothe affinity agent indicates the presence of anthrax exposure andinfection in the animal.

Anti-Bacillus-anthracis antibodies may be detected before the onset ofsymptoms in the animal. In one embodiment of the invention, the anti-B.anthracis antibodies may be detected at least four days after exposureto anthrax. In another embodiment, the anti-B. anthracis antibodies maybe detected up to 7 days after exposure to anthrax. In anotherembodiment, the anti-B. anthracis antibodies may be detected up to 14days after exposure to anthrax. In yet another embodiment, the anti-B.anthracis antibodies may be detected between 4 and 14 days afterexposure to anthrax. In another aspect, the anti-B. anthracis antibodiesmay be detected greater than 14 days after exposure to anthrax.

Biological samples to test for the presence of anthrax, i.e. testing forthe presence of antibodies specific for an epitope of B. anthracis ortesting for antigenic determinants of B. anthracis, can be collectedusing known methods. The sample can be taken directly from an animal orit can be in partially purified form or purified form. In one embodimentof the invention, blood drawn from a human is tested. In anotherembodiment, lymph fluids are tested.

VI. Capture Reagents

The present invention provides capture reagents that are capable ofspecifically binding SAP. Capture reagents can specifically bind SAPpolypeptides, fragments of SAP polypeptides or polypeptides containingone or more SAP epitopes. Capture reagents can be antibodies specificfor Bacillus anthracis. In addition, capture reagents are useful foridentifying affinity agents that specifically bind to anti-SAPantibodies.

The present invention, therefore, employs antibodies to B. anthracis ascapture reagents that specifically bind to B. anthracis epitopes in asample. Capture reagents of the invention can be obtained, for example,using a B. anthracis SAP polypeptide as an immunogen. The entire SAP canbe used as a capture reagent, or polypeptide subfragments that includean immunogenic epitope of SAP can be used. Suitable SAP polypeptides orfragments thereof can be isolated from B. anthracis, or more preferablycan be produced using recombinant methods.

The invention provides capture reagents that can specifically bind B.anthracis SAP polypeptides or fragments thereof. Capture reagents canalso be, for example, antibodies prepared using as immunogens, includingnatural, recombinant or synthetic polypeptides derived from B. anthracisSAP. The amino acid sequence of a B. anthracis SAP is shown as SEQ IDNO:1. Such polypeptides can function as immunogens that can be used forthe production of monoclonal or polyclonal antibodies. Immunogenicpeptides derived from SAP can also be used as immunogens; such peptidesare sometimes conjugated to a carrier polypeptide prior to inoculation.Immunogens can be used in either pure or impure form. Production ofantibodies against SAP polypeptides or polypeptides containing a SAPepitope is discussed in more detail below. Suitable capture reagentsalso include those that are obtained using methods such as phagedisplay.

Various procedures known in the art can be used for the production ofantibodies that specifically bind to a SAP epitope. For the productionof polyclonal antibodies, one can use SAP to inoculate any of varioushost animals, including but not limited to rabbits, mice, rats, sheep,goats, and the like. The SAP polypeptide can be prepared by recombinantmeans as described above using an expression vector containing a nucleicacid that encodes the B. anthracis SAP. For example, a nucleotidesequence encoding a B. anthracis SAP beginning at approximately 30 aminoacids from the published N-terminus (i.e., at the presumed cleavagesequence) is presented in SEQ ID NO:2.

Monoclonal antibodies can be prepared by any technique that provides forthe production of antibody molecules by continuous cell lines inculture, including the hybridoma technique originally developed byKohler and Milstein ((1975) Nature 256: 495-497), as well as the triomatechnique, the human B-cell hybridoma technique (Kozbor et al. (1983)Immunology Today 4: 72), and the EBV-hybridoma technique to producehuman monoclonal antibodies (Cole et al. (1985) in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonalantibodies also can be produced in germ-free animals as was described inPCT/US89/02545 (Publication No. WO8912690, published Dec. 12, 1989) andU.S. Pat. No. 5,091,512.

Fragments of antibodies are also useful as capture reagents. Whilevarious antibody fragments can be obtained by the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term “antibody,” as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv). Single chain antibodies are alsouseful to construct detection moieties. Methods for producing singlechain antibodies were described in, for example, U.S. Pat. No.4,946,778. Techniques for the construction of Fab expression librarieswere described by Huse et al. (1989) Science 246: 1275-1281; thesetechniques facilitate rapid identification of monoclonal Fab fragmentswith the desired specificity for SAP. Suitable capture reagents alsoinclude those that are obtained using methods such as phage display.

To prepare a suitable antigen preparation, one can prepare an expressionlibrary from B. anthracis and screen the library with a polyclonalantibody that is raised against a crude preparation of SAP. The insertsfrom those expression plasmids that express the SAP are then subclonedand sequenced. The SAP-encoding inserts are cloned into an expressionvector and used to transform E. coli or other suitable host cells. Theresulting preparation of recombinant SAP is then used to inoculate ananimal, e.g., a mouse.

In one embodiment, the capture reagents are recombinantly producedpolyclonal or monoclonal antibodies that bind to SAP. Recombinantantibodies are typically produced by immunizing an animal with SAP,obtaining RNA from the spleen or other antibody-expressing tissue of theanimal, making cDNA, amplifying the variable domains of the heavy andlight immunoglobulin chains, cloning the amplified DNA into a phagedisplay vector, infecting E. coli, expressing the phage display library,and selecting those library members that express an antibody that bindsto SAP. Methods suitable for carrying out each of these steps aredescribed in, for example U.S. Pat. No. 6,057,098. In anotherembodiment, the antibody or other binding peptides are expressed on thesurface of a replicable genetic unit, such as a filamentous phage, andespecially phage M13, Fd and F1. Most work has inserted librariesencoding polypeptides to be displayed with either pIII or pVIII of thesephage, forming a fusion protein which is displayed on the surface of thephage. See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty,WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO 92/18619 (geneVIII). In one embodiment, the genes that encode the heavy and lightchains of antibodies present in the cDNA library are amplified using aset of primers that can amplify substantially all of the different heavyand light chains. The resulting amplified fragments that result from theamplification step are pooled and subjected to asymmetric PCR so thatonly one strand (e.g., the antisense strand) is amplified. The singlestrand products are phosphorylated, annealed to a single-stranded uraciltemplate (e.g., the vector BS45, described in U.S. Pat. No. 6,057,098,which has coding regions for the constant regions of mouse heavy andlight chains), and introduced into a uracil DNA glycosylase+host cell toenrich for vectors that contain the coding sequences for heavy and lightchain variable domains.

To screen for phage that express an antibody that binds to SAP, one canattach a label to SAP using methods known to those of skill in the art.In one aspect of the invention, the phage that display such antibodiesare selected using SAP to which is attached an immobilizable tag, e.g.,biotin. The phage are contacted with the biotinylated antigen, afterwhich the phage are selected by contacting the resulting complex withavidin attached to a magnetic latex bead or other solid support. Theselected phage are then plated, and may be screened with SAP to which isattached a detectable label.

In one embodiment, the library is enriched for those phage that displaymore than one antibody that binds to SAP. Methods and vectors that areuseful for this enrichment are described in U.S. Pat. No. 6,057,098. Thepanning can be repeated one or more times to enhance the specificity andsensitivity of the resulting antibodies. Preferably, panning iscontinued until the percentage of functional positives is at least about70%, more preferably at least about 80%, and most preferably at leastabout 90%.

A recombinant anti-SAP monoclonal antibody can then be selected byamplifying antibody-encoding DNA from individual plaques, cloning theamplified DNA into an expression vector, and expressing the antibody ina suitable host cell (e.g., E. coli). The antibodies are then tested forability to bind SAP.

Recombinant polyclonal antibodies are used because of the various formsof SAP that may be found in clinical samples due to, for example,proteolysis. The diverse fine binding specificity of members of apopulation of polyclonal antibodies often allows the population to bindto several forms of SAP (e.g., species variants, escape mutant forms,proteolytic fragments) to which a monoclonal reagent may be unable tobind. Methods for producing recombinant polyclonal antibodies aredescribed in U.S. Pat. No. 6,057,098. Specific methods of producingrecombinant polyclonal antibodies that bind to SAP are described in theExamples below.

Polyclonal antibodies can be prepared as described above, except that anindividual antibody is not selected. The phage may be enriched for thosethat display more than one copy of the respective antibodies. The phageare then selected for those that bind to SAP. For example, one can use abiotinylated anti-SAP monoclonal antibody and SAP to concentrate thosephage that express antibodies that bind to SAP. The biotinylatedmonoclonal antibody is immobilized on a solid support (e.g., magneticlatex) to which is attached avidin. The phage that are bound to theimmobilized SAP are eluted, plated, and the panning repeated until thedesired percentage of functional positives is obtained.

Once the capture reagents of this invention are produced, they can beused to detect SAP present in a biological sample.

VII. Detecting SAP Antigenic Determinants or SAP Polypeptides with aCapture Reagent

As well as providing a method for detecting antibodies specific for B.anthracis in a biological sample, the present invention also providesmethods for the detection of SAP in a sample.

Biological samples to test for the presence of SAP in the sample arecollected using the same methods for the collection of biologicalsamples to test for the presence of antibodies specific for SAP in thesample. The sample can be taken directly from an animal or it can be inpartially purified form or purified form. In one embodiment of theinvention, the biological sample collected is fractionated into twosamples. One sample is used to test for the presence of SAP in thesample and the other is used to test for the presence of antibodiesspecific for SAP in the sample.

In order to detect SAP in a sample, the present invention providescontacting the biological sample with a capture reagent, e.g.,antibodies to a B. anthracis surface array protein, under suitablereaction conditions. If a B. anthracis surface array protein or epitopesthereof are present in the sample, a complex of protein and capturereagent will form. Formation of a complex indicates that the animal fromwhich the sample was obtained was exposed to anthrax.

The present invention can detect B. anthracis in a biological samplewhen present in the sample at a concentration of about 10⁴ cfu/ml orless. Preferably, the detection limit for B. anthracis will be about5×10³ cfu/ml or less, more preferably about 1.8×10³ cfu/ml or less, andstill more preferably about 10³ cfu/ml or less.

SAP polypeptides or SAP antigenic determinants present in a sample canbe detected by the methods of the invention before the onset of symptomsin the animal. In one embodiment of the invention, the SAP polypeptidesor SAP antigenic determinants may be detected one day after exposure toanthrax. In another embodiment, the SAP polypeptides or SAP antigenicdeterminants may be detected up to 7 days after exposure to anthrax. Inanother embodiment, the SAP polypeptides or SAP antigenic determinantsmay be detected up to 14 days after exposure to anthrax. In yet anotherembodiment, the SAP polypeptides or SAP antigenic determinants may bedetected greater than 14 days after exposure to anthrax.

The methods of the present invention employ different immunologictechniques and immunoassays to detect B. anthracis SAP in a sample. TheB. anthracis detection methods of the present invention, like the B.anthracis antibody detection methods, can be carried out in a widevariety of assay formats. In one embodiment, the assay methods involveimmobilization of a capture reagent for B. anthracis SAP on a solidsupport, followed by detection of the immobilized or bound SAP. Thedetectable labels can be detected directly after immobilization on thesolid support, for example, or indirectly by an enzymatic or otherreaction that results in a detectable change in a reactant that ispresent in the detection assay reaction.

One method of detection is based on the ELISA method. See, e.g., Elderet al., J. Clin. Microbiol. 16:141 (1982); Ausubel et al., supra.Generally, antigens or capture reagents for antigens are fixed to asolid surface. Bound antigens are detected using antigen-specificantibodies that are detected by way of an enzymatic reaction. In oneembodiment, the ELISA method used is the “sandwich” method wherein theantigens are bound to the solid surface via capture reagent bound to thesolid surface. An antibody, or other antigen detection reagent,typically linked to an enzyme, is then contacted to the antigen, washed,then contacted with the enzyme substrate to select binding. These andother embodiments of the ELISA method are taught in, for example,Ausubel et al. § 11.2, supra.

To immobilize SAP on the solid support, a capture reagent thatspecifically binds to SAP is non-diffusively associated with thesupport. The capture reagents can be immobilized on the support eitherby covalent or non-covalent methods, which are known to those of skillin the art. See, e.g., Pluskal et al. (1986) BioTechniques 4: 272-283.Suitable supports include, for example, glasses, plastics, polymers,metals, metalloids, ceramics, organics, and the like. Specific examplesinclude, but are not limited to, microtiter plates, nitrocellulosemembranes, nylon membranes, and derivatized nylon membranes, beads, andalso particles, such as agarose, SEPHADEX™, and the like. Assay systemsfor use in the methods and kits of the invention include, but are notlimited to, dipstick-type devices, immunochromatographic test strips andradial partition immunoassay devices, microtiter assays and flow-throughdevices. Conveniently, where the solid support is a membrane, the testsample can flow through the membrane, for example, by gravity, capillaryaction, or under positive or negative pressure.

Once the sample has been contacted with the solid support, the solidsupport is then contacted with detection reagents for SAP. The solidsupport can be washed prior to contact with detection reagents to removeunbound reagents and test sample components. After incubation of thedetection reagents for a sufficient time to bind a substantial portionof the immobilized SAP, any unbound labeled reagents are removed by, forexample, washing. The detectable label associated with the detectionreagents is then detected. For example, in the case of an enzyme used asa detectable label, a substrate for the enzyme that turns a visiblecolor upon action of the enzyme is placed in contact with the bounddetection reagent. A visible color will then be observed in proportionto the amount of the specific antigen in the sample.

Other detection systems, such as those described for detecting antibodywith an affinity agent, for e.g., Western blotting, can be adapted todetect antigen with a capture reagent, including membrane-baseddetection methods. Any of the assays described herein can be used toconfirm the results of another assay.

VIII. Detection Reagents

As discussed above, the presence of SAP can be detected using adetection reagent that is composed of a binding moiety that specificallybinds to SAP. In addition, anti-SAP antibodies in a biological samplespecific for SAP are generally detected using an antibody, or othercapture reagent, that specifically binds to the anti-SAP antibodies, orcomplex of anti-SAP antibodies and affinity agent, in the sample. Thedetection reagents are either directly labeled, i.e., comprise or reactto produce a detectable label, or are indirectly labeled, i.e., bind toa molecule that is itself labeled with a detectable label. Labels can bedirectly attached to or incorporated into the detection reagent bychemical or recombinant methods.

In one embodiment, a label is coupled to a molecule, such as an antibodythat specifically binds to SAP, through a chemical linker. In anotherembodiment, a label is coupled to an antibody that specifically binds tohuman antibodies to SAP. Linker domains are typically polypeptidesequences, such as poly-gly sequences of between about 5 and 200 aminoacids. In some embodiments, proline residues are incorporated into thelinker to prevent the formation of significant secondary structuralelements by the linker. Linkers may be flexible amino acid subsequencesthat are synthesized as part of a recombinant fusion protein comprisingthe RNA recognition domain. In one embodiment, the flexible linker is anamino acid subsequence that includes a proline, such asGly(x)-Pro-Gly(x) where x is a number between about 3 and about 100. Inother embodiments, a chemical linker is used to connect synthetically orrecombinantly produced recognition and labeling domain subsequences.Such flexible linkers are known to persons of skill in the art. Forexample, poly(ethylene glycol) linkers are available from ShearwaterPolymers, Inc. Huntsville, Ala. These linkers optionally have amidelinkages, sulfhydryl linkages, or heterofunctional linkages.

The detectable labels used in the assays of the present invention, whichare attached to the antibodies, can be primary labels (where the labelcomprises an element that is detected directly or that produces adirectly detectable element) or secondary labels (where the detectedlabel binds to a primary label, e.g., as is common in immunologicallabeling). An introduction to labels, labeling procedures and detectionof labels is found in Polak and Van Noorden (1997) Introduction toImmunocytochemistry, 2nd ed., Springer Verlag, NY and in Haugland (1996)Handbook of Fluorescent Probes and Research Chemicals, a combinedhandbook and catalogue Published by Molecular Probes, Inc., Eugene,Oreg. patents that described the use of such labels include U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;and 4,366,241.

Primary and secondary labels can include undetected elements as well asdetected elements. Useful primary and secondary labels in the presentinvention can include spectral labels such as green fluorescent protein,fluorescent dyes (e.g., fluorescein and derivatives such as fluoresceinisothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives(e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.),digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase etc.), spectralcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. The label can becoupled directly or indirectly to a component of the detection assay(e.g., the detection reagent) according to methods well known in theart. As indicated above, a wide variety of labels may be used, with thechoice of label depending on sensitivity required, ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions.

Labels include those that use: 1) chemiluminescence (using horseradishperoxidase and/or alkaline phosphatase with substrates that producephotons as breakdown products as described above) with kits beingavailable, e.g., from Molecular Probes, Amersham, Boehringer-Mannheim,and Life Technologies/Gibco BRL; 2) color production (using bothhorseradish peroxidase and/or alkaline phosphatase with substrates thatproduce a colored product (kits available from Life Technologies/GibcoBRL, and Boehringer-Mannheim)); 3) fluorescence using, e.g., an enzymesuch as alkaline phosphatase, together with the substrate AttoPhos(Amersham) or other substrates that produce fluorescent products, 4)fluorescence (e.g., using Cy-5 (Amersham), fluorescein, and otherfluorescent tags); 5) radioactivity. Other methods for labeling anddetection will be readily apparent to one skilled in the art.

For use of the present invention outside the laboratory, labels arenon-radioactive and readily detected without the necessity ofsophisticated instrumentation. Preferably, detection of the labels willyield a visible signal that is immediately discernable upon visualinspection. One example of detectable secondary labeling strategies usesan antibody that recognizes SAP in which the antibody is linked to anenzyme (typically by recombinant or covalent chemical bonding). Theantibody is detected when the enzyme reacts with its substrate,producing a detectable product. Enzymes that can be conjugated todetection reagents of the invention include, e.g., β-galactosidase,luciferase, horse radish peroxidase, and alkaline phosphatase. Thechemiluminescent substrate for luciferase is luciferin. One embodimentof a fluorescent substrate for β-galactosidase is4-methylumbelliferyl-β-D-galactoside. Embodiments of alkalinephosphatase substrates include p-nitrophenyl phosphate (pNPP), which isdetected with a spectrophotometer; 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TRphosphate, which are detected visually; and4-methoxy-4-(3-phosphonophenyl) spiro[1,2-d]oxetane-3,2′-adamantane],which is detected with a luminometer. Embodiments of horse radishperoxidase substrates include 2,2′azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, ando-phenylenediamine (OPD), which are detected with a spectrophotometer;and 3,3,5,5′-tetramethylbenzidine (TMB), 3,3′diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N), whichare detected visually. Other suitable substrates are known to thoseskilled in the art. The enzyme-substrate reaction and product detectionare performed according to standard procedures known to those skilled inthe art and kits for performing enzyme immunoassays are available asdescribed above.

The presence of a label can be detected by inspection, or a detectorwhich monitors a particular probe or probe combination can be used todetect the detection reagent label. Typical detectors includespectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

IX. Kits for the Detection of SAP

This invention also provides kits for the detection and/orquantification of anthrax using the methods described herein. The kitscan include a container containing one or more of the above-discussedreagents with or without labels, either free or bound to solid supports.A suitable solid support, such as a membrane, can also be included inthe kits of the invention. The kits can provide solid supports in theform of an assay apparatus that is adapted to use in the describedassay. Preferably, the kits will also include reagents used in thedescribed assays, including reagents useful for detecting the presenceof the detectable labels. Other materials useful in the performance ofthe assays can also be included in the kits, including test tubes,transfer pipettes, and the like. The kits can also include writteninstructions for the use of one or more of these reagents in any of theassays described herein.

The kits of the invention can also include an internal and/or anexternal control. An internal control can consist of the SAP polypeptideor an anti-SAP antibody. The control antigen can conveniently bepreattached to a capture reagent in a zone of the solid support adjacentto the zone to which the sample is applied. The external control canalso consist of a SAP polypeptide or an anti-SAP antibody. In someembodiments, the antigen present in the external control will be at aconcentration at or above the sensitivity limit of the assay means. Theexternal control antigen can be diluted in the sample diluent andassayed in the same manner as would a biological sample. Alternatively,the external control SAP polypeptide or anti-SAP antibody can be addedto an aliquot of an actual biological sample to determine thesensitivity of the assay. The kits of the present invention can containmaterial sufficient for one assay, or can contain sufficient materialsfor multiple assays.

All publications cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

EXAMPLES

The following examples are offered to illustrate, but not to limit thepresent invention.

Example 1 Isolation of a Gene Encoding Bacillus anthracis Surface ArrayProtein (SAP)

This Example describes the cloning and characterization of a gene thatencodes a Bacillus anthracis surface array protein (SAP).

Isolation of B. anthracis DNA

Bacillus anthracis genomic DNA isolated from the non-pathologic Sternestrain was used as a template source for PCR amplification of a nucleicacid that codes for SAP. A total of 6 ml of Bacillus anthracis Sternestrain (1×10¹⁰/ml in PBS pH 7.4) was pelleted in a microcentrifuge at10,000 g for 5 minutes. Bacterial pellets were then combined andresuspended in a final volume of 1 ml lysis buffer (50 mMTris(hydroxymethyl) aminomethane (“Tris”) pH 7.8, 10 mMethylenediaminetetraacetic acid (“EDTA”), 100 μg/ml Ribonuclease (RNaseA) (Roche Molecular Biochemical, Indianapolis, Ind.), 0.5% Triton X-100M(T-octylphenoxypolyethoxyethanol) (Sigma, St. Louis, Mo.), 12.5%sucrose). Lysozyme (Sigma) was added to a final concentration of 2 mg/mland the mixture incubated for 1 hr at 37° C. 300 μg of Proteinase K(Roche Molecular Biochemical, Indianapolis, Ind.) and a one-tenth volumeof 10% SDS was added to the mixture followed by a 1 hr incubation at 56°C. NaCl was then added to a final concentration of 500 mM by addingone-tenth volume of 5 M NaCl. The mixture was then twice extracted withphenol/chloroform (phenol:chloroform:isoamyl alcohol (50:49:1)) and theDNA in the aqueous layer sheared by passing the solution through an 18gauge needle. DNA was precipitated with 2.5 volumes of ethanol andresuspended in 200 μl of distilled water. This DNA preparation wasextracted once with Tris pH 8 equilibrated phenol, two times withphenol/chloroform and finally twice with chloroform (chloroform:isoamylalcohol (49:1)) alone. DNA in the aqueous layer was precipitated a finaltime and resuspended in 500 μl of distilled water, yieldingapproximately 79 μg of DNA at 158 μg/ml. This DNA was used as a templatein the subsequent PCR amplification of the SAP gene.

Cloning of Bacillus anthracis Sap Gene Via PCR

Appropriate PCR primers were made corresponding to the coding sequenceof the 5′ and 3′ ends of the B. anthracis SAP gene (see primer sequencebelow). These primers were based on a published nucleotide sequence(Etienne-Toumelin et al., supra). DNA encoding the native signalsequence of SAP (amino acids 1-29) was purposefully omitted from thecloning since a functional signal sequence was provided by theexpression vector pBRncoH3 (described in copending, commonly-owned U.S.patent application Ser. No. 08/835,159, filed Apr. 4, 1997). The 5′primer contains 23 bases of vector sequence at its 5′-end thatcorresponds to the 3′-end of the pBRncoH3 vector. The 3′ primer contains19 bases of the tetracycline promoter, removed by HindIII digestion inthe vector, in addition to 20 bases of vector sequence 3′ to the HindIIIsite. The 3′ primer was also engineered to encode a hexahistidine aminoacid tag at the C-terminus of the SAP protein to allow for efficientpurification using nickel-chelate affinity chromatography (see below).

5′ PCR primer: (SEQ ID NO:2) 5′-TCGCTGCCCAACCAGCCATGGCCGCAGGTAAAACATTCCCA GAC -3′ 3′ PCR primer: (SEQ IDNO:3) 5′- GTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAATTAGTGATGGTGATGGTGATGTTTTGTTGCAGGTTTTGCTTCTTT -3′

The nucleic acid that encodes SAP was amplified using these primers andapproximately 30 ng of Bacillus anthracis genomic DNA as template. Theamplification was performed using Expand™ DNA polymerase (RocheMolecular Biochemical (Indianapolis, Ind.). SAP insert DNA (˜300 ng) waspurified and annealed to the HindIII-digested pBRncoH3 vector (100 ng)at a 6:1 molar ratio of insert to vector. An aliquot was electroporatedinto 40 μl of electrocompetent E. coli strain DH10B as described inExample 3. Various dilutions of the transformed cells were plated on LBagar plates supplemented with tetracycline (10 μg/ml) and grownovernight at 37° C. Three colonies were each picked into 3 ml 2×YT,supplemented with tetracycline (10 μg/ml), and grown overnight at 37° C.The following day, glycerol freezer stocks were made for long termstorage at −80° C.

In order to confirm that the SAP gene had indeed been cloned, each ofthe three clones was tested for the ability to synthesize SAP proteinupon arabinose induction as described below. All three clones produced aprotein of the predicted size, approximately 94 kDa in molecular mass,and were shown to react with a rabbit anti-anthracis polyclonal serumusing Western blot analysis (data not shown). Two of the three cloneswere sequenced and compared against the National Center forBiotechnology Information's (NCBI) non-redundant nucleotide databaseusing the BLAST search engine. This search indicated that a SAP gene hadindeed been cloned. There were eight differences in the predicted aminoacid sequence compared to the noted published sequence. These changesare lysine 264 to arginine, glutamic acid 478 to alanine, arginine 482to histidine, glutamic acid 496 to aspartic acid, lysine 556 toarginine, glutamic acid 606 to aspartic acid, lysine 607 to threonine,and valine 751 to alanine. Amino acid numbering is based on thepublished sequence (Etienne-Toumelin et al., supra). These differencesmay be due to the fact that a different Bacillus anthracis strain wasused in the work described here. The original published work did not usethe Sterne strain. The predicted amino acid sequence of the SAP genecloned here shows 8 amino acid differences out of 785, and is thus 99.0%identical to the published sequence.

Example 2 Expression and Purification of Recombinant Bacillus anthracisSAP from E. coli

This Example describes the expression and purification of B. anthracisSAP using E. coli.

A shake flask containing 2×YT supplemented with 1% glycerol wasinoculated with an E. coli DH10B strain from Example 1 that contained acloned B. anthracis SAP gene and incubated overnight in an Innova 4330incubator shaker (New Brunswick Scientific, Edison, N.J.) set at 37° C.,300 rpm. The inoculum was used to seed 500 mL cultures of defined medium(Pack et al. (1993) Bio/Technology 11: 1271-1277) supplemented with 3g/L L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco, Detroit,Mich.), 12.5 g/L glycerol and 10 μg/ml tetracycline. Cultures were grownin 2 L Tunair shake flasks (Shelton Scientific, Shelton, Conn.) at 37°C. and 300 rpm. Cells were grown to an optical density of approximately4 absorption units at 600 nm. Expression of SAP was then induced byaddition of L(+)-arabinose to 2 g/L during this logarithmic growthphase. The flasks were then maintained at 23° C. and 300 rpm overnight.

The following morning, bacterial cultures were passed through an M-110YMicrofluidizer (Microfluidics, Newton, Mass.) at 17,000 psi. Thehomogenate was clarified in a J2-21 centrifuge (Beckman, Fullerton,Calif.) and recombinant SAP purified from the supernatant usingimmobilized metal affinity chromatography. Briefly, Chelating SepharoseFastFlow™ resin (Pharmacia, Piscataway, N.J.) was charged with 0.1 MNiCl₂ and equilibrated in 20 mM borate, 150 mM NaCl, 10 mM imidazole,0.01% NaN₃, pH 8. A stock solution was used to bring the supernatantconcentration to 10 mM imidazole, pH 8. Chelating resin was then addedto the supernatant and the mixture shaken for 1 hour at roomtemperature, 150-200 rpm. During this time, SAP was captured by means ofthe high affinity interaction between nickel and the hexahistidine tagengineered onto the C-terminus of SAP. After 1 hour, the resin mixturewas poured into a chromatography column and washed with 20 mM borate,150 mM NaCl, 10 mM imidazole, 0.01% NaN₃, pH 8.0. SAP was eluted fromthe resin with the same buffer containing 200 mM imidazole instead of 10mM.

The volume of eluted SAP was reduced using a centrifuge concentratorwith a 30 kDa molecular weight cut off (Amicon, Beverly, Mass.), and thesample subsequently dialyzed against sterile phosphate-buffered solution(PBS) for immunizations and BBS (20 mM borate, 150 mM NaCl, 0.01% NaN₃,pH 8.0) for biotinylation. Isolated SAP was evaluated for purity bySDS-PAGE analysis and shown to be greater than 95% pure. The proteinconcentration of recombinant SAP was determined by UV absorbance at 280nm, assuming an absorbance of 0.593 for a 1 mg/ml solution.

Example 3 Construction of a Phage-Display Library

This Example describes the construction of a phage display library fromwhich binding reagents that are specific for B. anthracis SAP wereidentified.

Immunization and mRNA Isolation

A phage display library for identification of SAP-binding molecules wasconstructed as follows. A/J mice (Jackson Laboratories, Bar Harbor, Me.)were immunized intraperitoneally with recombinant SAP antigen, using 100μg protein in Freund's complete adjuvant, on day 0, and with 100 μgantigen on day 28. Test bleeds of mice were obtained through puncture ofthe retro-orbital sinus. If, by testing the titers, they were deemedhigh by ELISA using biotinylated SAP antigen immobilized via neutravidin(Reacti-Bind™ NeutrAvidin™-Coated Polystyrene Plates, Pierce, Rockford,Ill.), the mice were boosted with 100 μg of protein on day 70, 71 and72, with subsequent sacrifice and splenectomy on day 77. If titers ofantibody were not deemed satisfactory, mice were boosted with 100 μgantigen on day 56 and a test bleed taken on day 63. If satisfactorytiters were obtained, the animals were boosted with 100 μg of antigen onday 98, 99, and 100 and the spleens harvested on day 105.

The spleens were harvested in a laminar flow hood and transferred to apetri dish, trimming off and discarding fat and connective tissue. Thespleens were macerated quickly with the plunger from a sterile 5 ccsyringe in the presence of 1.0 ml of solution D (25.0 g guanidinethiocyanate (Boehringer Mannheim, Indianapolis, Ind.), 29.3 ml sterilewater, 1.76 ml 0.75 M sodium citrate pH 7.0, 2.64 ml 10% sarkosyl(Fisher Scientific, Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol (FisherScientific, Pittsburgh, Pa.)). This spleen suspension was pulled throughan 18 gauge needle until all cells were lysed and the viscous solutionwas transferred to a microcentrifuge tube. The petri dish was washedwith 100 μl of solution D to recover any remaining spleen. Thissuspension was then pulled through a 22 gauge needle an additional 5-10times.

The sample was divided evenly between two microcentrifuge tubes and thefollowing added, in order, with mixing by inversion after each addition:50 μl 2 M sodium acetate pH 4.0, 0.5 ml water-saturated phenol (FisherScientific, Pittsburgh, Pa.), 100 μl chloroform/isoamyl alcohol 49:1(Fisher Scientific, Pittsburgh, Pa.). The solution was vortexed for 10seconds and incubated on ice for 15 min. Following centrifugation at 14krpm for 20 min at 2-8° C., the aqueous phase was transferred to a freshtube. An equal volume of water saturated phenol:chloroform:isoamylalcohol (50:49:1) was added, and the tube vortexed for ten seconds.After a 15 min incubation on ice, the sample was centrifuged for 20 minat 2-8° C., and the aqueous phase transferred to a fresh tube andprecipitated with an equal volume of isopropanol at −20° C. for aminimum of 30 min. Following centrifugation at 14 krpm for 20 min at 4°C., the supernatant was aspirated away, the tubes briefly spun and alltraces of liquid removed from the RNA pellet.

The RNA pellets were each dissolved in 300 μl of solution D, combined,and precipitated with an equal volume of isopropanol at −20° C. for aminimum of 30 min. The sample was centrifuged 14 krpm for 20 min at 4°C., the supernatant aspirated as before, and the sample rinsed with 100μl of ice-cold 70% ethanol. The sample was again centrifuged 14 krpm for20 min at 4° C., the 70% ethanol solution aspirated, and the RNA pelletdried in vacuo. The pellet was resuspended in 100 μl of sterile diethylpyrocarbonate-treated water. The concentration was determined by A₂₆₀using an absorbance of 1.0 for a concentration of 40 μg/ml. The RNAswere stored at −80° C.

Preparation of Complementary DNA (cDNA)

The total RNA purified from mouse spleens as described above was useddirectly as template for cDNA preparation. RNA (50 μg) was diluted to100 μL with sterile water, and 10 μL of 130 ng/μL oligo dT₁₂(synthesized on Applied Biosystems Model 392 DNA synthesizer) was added.The sample was heated for 10 min at 70° C., then cooled on ice. Forty μL5× first strand buffer was added (Gibco/BRL, Gaithersburg, Md.), alongwith 20 μL 0.1 M dithiothreitol (Gibco/BRL, Gaithersburg, Md.), 10 μL 20mM deoxynucleoside triphosphates (dNTP's, Boehringer Mannheim,Indianapolis, Ind.), and 10 μL water on ice. The sample was thenincubated at 37° C. for 2 min. Ten μL reverse transcriptase(Superscript™ II, Gibco/BRL, Gaithersburg, Md.) was added and incubationwas continued at 37° C. for 1 hr. The cDNA products were used directlyfor polymerase chain reaction (PCR).

Amplification of Antibody Genes by PCR

To amplify substantially all of the H and L chain genes using PCR,primers were chosen that corresponded to substantially all publishedsequences. Because the nucleotide sequences of the amino termini of Hand L contain considerable diversity, 33 oligonucleotides weresynthesized to serve as 5′ primers for the H chains, and 29oligonucleotides were synthesized to serve as 5′ primers for the kappa Lchains as described in U.S. patent application Ser. No. 08/835,159,filed Apr. 4, 1997. The constant region nucleotide sequences for eachchain required only one 3′ primer for the H chains and one 3′ primer forthe kappa L chains.

A 50 μL reaction was performed for each primer pair with 50 μmol of 5′primer, 50 μmol of 3′ primer, 0.25 μL Taq DNA Polymerase (5 units/μL,Boehringer Mannheim, Indianapolis, Ind.), 3 μL cDNA (prepared asdescribed in Example 3), 5 μL 2 mM dNTP's, 5 μL 10×Taq DNA polymerasebuffer with MgCl₂ (Boehringer Mannheim, Indianapolis, Ind.), and H₂O to50 μL. Amplification was done using a GeneAmp® 9600 thermal cycler(Perkin Elmer, Foster City, Calif.) with the following thermocycleprogram: 94° C. for 1 min; 30 cycles of 94° C. for 20 sec, 55° C. for 30sec, and 72° C. for 30 sec; 72° C. for 6 min; 4° C.

The dsDNA products of the PCR process were then subjected to asymmetricPCR using only a 3′ primer to generate substantially only the anti-sensestrand of the target genes. A 100 μL reaction was done for each dsDNAproduct with 200 μmol of 3′ primer, 2 μL of ds-DNA product, 0.5 μL TaqDNA Polymerase, 10 μL 2 mM dNTP's, 10 μL 10×Taq DNA polymerase bufferwith MgCl₂ (Boehringer Mannheim, Indianapolis, Ind.), and H₂O to 100 μL.The same PCR program as that described above was used to amplify thesingle-stranded (ss)-DNA.

Purification of Single-Stranded DNA by High Performance LiquidChromatography and Kinasing Single-Stranded DNA

The H chain ss-PCR products and the L chain single-stranded PCR productswere ethanol precipitated by adding 2.5 volumes ethanol and 0.2 volumes7.5 M ammonium acetate and incubating at −20° C. for at least 30 min.The DNA was pelleted by centrifuging in an Eppendorf centrifuge at 14krpm for 10 min at 2-8° C. The supernatant was carefully aspirated, andthe tubes were briefly spun a 2nd time. The last drop of supernatant wasremoved with a pipette. The DNA was dried in vacuo for 10 min on mediumheat. The H chain products were pooled in 210 μL water and the L chainproducts were pooled separately in 210 μL water. The single-stranded DNAwas purified by high performance liquid chromatography (HPLC) using aHewlett Packard 1090 HPLC and a Gen-Pak™ FAX anion exchange column(Millipore Corp., Milford, Mass.). The gradient used to purify thesingle-stranded DNA is shown in Table 1, and the oven temperature was60° C. Absorbance was monitored at 260 nm. The single-stranded DNAeluted from the HPLC was collected in 0.5 min fractions. Fractionscontaining single-stranded DNA were ethanol precipitated, pelleted anddried as described above. The dried DNA pellets were pooled in 200 μLsterile water.

TABLE 1 HPLC gradient for purification of ss-DNA Time (min) % A % B % CFlow (ml/min) 0 70 30 0 0.75 2 40 60 0 0.75 17 15 85 0 0.75 18 0 100 00.75 23 0 100 0 0.75 24 0 0 100 0.75 28 0 0 100 0.75 29 0 100 0 0.75 340 100 0 0.75 35 70 30 0 0.75 Buffer A is 25 mM Tris, 1 mM EDTA, pH 8.0Buffer B is 25 mM Tris, 1 mM EDTA, 1 M NaCl, pH 8.0 Buffer C is 40 mmphosphoric acid

The single-stranded DNA was 5′-phosphorylated in preparation formutagenesis. Twenty-four μL 10× kinase buffer (United StatesBiochemical, Cleveland, Ohio), 10.4 μL 10 mM adenosine-5′-triphosphate(Boehringer Mannheim, Indianapolis, Ind.), and 2 μL polynucleotidekinase (30 units/μL, United States Biochemical, Cleveland, Ohio) wasadded to each sample, and the tubes were incubated at 37° C. for 1 hr.The reactions were stopped by incubating the tubes at 70° C. for 10 min.The DNA was purified with one extraction of Tris equilibrated phenol(pH>8.0, United States Biochemical, Cleveland, Ohio):chloroform:isoamylalcohol (50:49:1) and one extraction with chloroform:isoamyl alcohol(49:1). After the extractions, the DNA was ethanol precipitated andpelleted as described above. The DNA pellets were dried, then dissolvedin 50 μL sterile water. The concentration was determined by measuringthe absorbance of an aliquot of the DNA at 260 nm using 33 μg/ml for anabsorbance of 1.0. Samples were stored at −20° C.

Preparation of Uracil Templates Used in Generation of Spleen AntibodyPhage Libraries

One ml of E. coli CJ236 (BioRAD, Hercules, Calif.) overnight culture wasadded to 50 ml 2×YT in a 250 ml baffled shake flask. The culture wasgrown at 37° C. to OD₆₀₀=0.6, inoculated with 10 μl of a 1/100 dilutionof BS45 vector phage stock (described in U.S. patent application Ser.No. 08/835,159, filed Apr. 4, 1997) and growth continued for 6 hr.Approximately 40 ml of the culture was centrifuged at 12 krpm for 15minutes at 4° C. The supernatant (30 ml) was transferred to a freshcentrifuge tube and incubated at room temperature for 15 minutes afterthe addition of 15 μl of 10 mg/ml RNaseA (Boehringer Mannheim,Indianapolis, Ind.). The phage were precipitated by the addition of 7.5ml of 20% polyethylene glycol 8000 (Fisher Scientific, Pittsburgh,Pa.)/3.5M ammonium acetate (Sigma Chemical Co., St. Louis, Mo.) andincubation on ice for 30 min. The sample was centrifuged at 12 krpm for15 min at 2-8° C. The supernatant was carefully discarded, and the tubebriefly spun to remove all traces of supernatant. The pellet wasresuspended in 400 μl of high salt buffer (300 mM NaCl, 100 mM Tris pH8.0, 1 mM EDTA), and transferred to a 1.5 ml tube.

The phage stock was extracted repeatedly with an equal volume ofequilibrated phenol:chloroform:isoamyl alcohol (50:49:1) until no traceof a white interface was visible, and then extracted with an equalvolume of chloroform:isoamyl alcohol (49:1). The DNA was precipitatedwith 2.5 volumes of ethanol and ⅕ volume 7.5 M ammonium acetate andincubated 30 min at −20° C. The DNA was centrifuged at 14 krpm for 10min at 4° C., the pellet washed once with cold 70% ethanol, and dried invacuo. The uracil template DNA was dissolved in 30 μl sterile water andthe concentration determined by A₂₆₀ using an absorbance of 1.0 for aconcentration of 40 μg/ml. The template was diluted to 250 ng/μL withsterile water, aliquoted, and stored at −20° C.

Mutagenesis of Uracil Template with ss-DNA and Electroporation into E.coli to Generate Antibody Phage Libraries

Antibody phage display libraries were generated by simultaneouslyintroducing single-stranded heavy and light chain genes onto a phagedisplay vector uracil template. A typical mutagenesis was performed on a2 μg scale by mixing the following in a 0.2 ml PCR reaction tube: 8 μlof (250 ng/μL) uracil template, 8 μL of 10× annealing buffer (200 mMTris pH 7.0, 20 mM MgCl₂, 500 mM NaCl), 3.33 μl of kinasedsingle-stranded heavy chain insert (100 ng/μL), 3.1 μl of kinasedsingle-stranded light chain insert (100 ng/μL), and sterile water to 80μl. DNA was annealed in a GeneAmp® 9600 thermal cycler using thefollowing thermal profile: 20 sec at 94° C., 85° C. for 60 sec, 85° C.to 55° C. ramp over 30 min, hold at 55° C. for 15 min. The DNA wastransferred to ice after the program finished. The extension/ligationwas carried out by adding 8 μl of 10× synthesis buffer (5 mM each dNTP,10 mM ATP, 100 mM Tris pH 7.4, 50 mM MgCl₂, 20 mM DTT), 8 μL T4 DNAligase (1 U/μL, Boehringer Mannheim, Indianapolis, Ind.), 8 μL dilutedT7 DNA polymerase (1 U/μL, New England BioLabs, Beverly, Mass.) andincubating at 37° C. for 30 min. The reaction was stopped with 300 μL ofmutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM EDTA). The mutagenesisDNA was extracted once with equilibrated phenol(pH>8):chloroform:isoamyl alcohol (50:49:1), once withchloroform:isoamyl alcohol (49:1), and the DNA was ethanol precipitatedat −20° C. for at least 30 min. The DNA was pelleted and the supernatantcarefully removed as described above. The sample was briefly spun againand all traces of ethanol removed with a pipetman. The pellet was driedin vacuo. The DNA was resuspended in 4 μL of sterile water.

One microliter of mutagenesis DNA (500 ng) was transferred into 40 μlelectrocompetent E. coli DH12S (Gibco/BRL, Gaithersburg, Md.) usingelectroporation. The transformed cells were mixed with approximately 1.0ml of overnight XL-1 cells which were diluted with 2×YT broth to 60% theoriginal volume. This mixture was then transferred to a 15-ml sterileculture tube and 9 ml of top agar added for plating on a 150-mm LB agarplate. Plates were incubated for 4 hrs at 37° C. and then transferred to20° C. overnight. First round antibody phage were made by eluting phageoff these plates in 10 ml of 2×YT, spinning out debris, and taking thesupernatant. These samples are the antibody phage display libraries usedfor selecting antibodies against SAP. Efficiency of the electroporationswas measured by plating 10 μl of a 10⁻⁴ dilution of suspended cells onLB agar plates, follow by overnight incubation of plates at 37° C. Theefficiency was calculated by multiplying the number of plaques on the10⁻⁴ dilution plate by 106. Library electroporation efficiencies aretypically greater than 1×10⁷ phage under these conditions.

Transformation of E. coli by Electroporation

Electrocompetent E. coli cells were thawed on ice. DNA was mixed with 40L of these cells by gently pipetting the cells up and down 2-3 times,being careful not to introduce an air bubble. The cells were transferredto a Gene Pulser cuvette (0.2 cm gap, BioRAD, Hercules, Calif.) that hadbeen cooled on ice, again being careful not to introduce an air bubblein the transfer. The cuvette was placed in the E. coli Pulser (BioRAD,Hercules, Calif.) and electroporated with the voltage set at 1.88 kVaccording to the manufacturer's recommendation. The transformed samplewas immediately resuspended in 1 ml of 2×YT broth or 1 ml of a mixtureof 400 μl 2×YT/600 μl overnight XL-1 cells and processed as proceduresdictated.

Plating M13 Phage or Cells Transformed with Antibody Phage-DisplayVector Mutagenesis Reaction

Phage samples were added to 200 μL of an overnight culture of E. coliXL1-Blue when plating on 100 mm LB agar plates or to 600 μL of overnightcells when plating on 150 mm plates in sterile 15 ml culture tubes.After adding LB top agar (3 ml for 100 mm plates or 9 ml for 150 mmplates, top agar stored at 55° C. (see, Appendix A1, Sambrook et al.,supra.), the mixture was evenly distributed on an LB agar plate that hadbeen pre-warmed (37° C.-55° C.) to remove any excess moisture on theagar surface. The plates were cooled at room temperature until the topagar solidified. The plates were inverted and incubated at 37° C. asindicated.

Preparation of Biotinylated Sap and Biotinylated Antibodies

Concentrated recombinant SAP antigen (Example 2 above) was extensivelydialyzed into BBS (20 mM borate, 150 mM NaCl, 0.1% NaN₃, pH 8.0). Afterdialysis, 1 mg of SAP (1 mg/ml in BBS) was reacted with a 15 fold molarexcess of biotin-XX-NHS ester (Molecular Probes, Eugene, Oreg., stocksolution at 40 mM in DMSO). The reaction was incubated at roomtemperature for 90 min and then quenched with taurine (Sigma ChemicalCo., St. Louis, Mo.) at a final concentration of 20 mM. The biotinylatedreaction mixture was then dialyzed against BBS at 2-8° C. Afterdialysis, biotinylated SAP was diluted in panning buffer (40 mM Tris,150 mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH 7.5), aliquoted, and storedat −80° C. until needed.

Antibodies were reacted with 3-(N-maleimidylpropionyl)biocytin(Molecular Probes, Eugene, Oreg.) using a free cysteine located at thecarboxy terminus of the heavy chain. Antibodies were reduced by addingDTT to a final concentration of 1 mM for 30 min at room temperature.Reduced antibody was passed through a Sephadex G50 desalting columnequilibrated in 50 mM potassium phosphate, 10 mM boric acid, 150 mMNaCl, pH 7.0. 3-(N-maleimidylpropionyl)-biocytin was added to a finalconcentration of 1 mM and the reaction allowed to proceed at roomtemperature for 60 min. Samples were then dialyzed extensively againstBBS and stored at 2-8° C.

Preparation of Avidin Magnetic Latex

The magnetic latex (Estapor, 10% solids, Bangs Laboratories, Fishers,Ind.) was thoroughly resuspended and 2 ml aliquoted into a 15 ml conicaltube. The magnetic latex was suspended in 12 ml distilled water andseparated from the solution for 10 min using a magnet (PerSeptiveBiosystems, Framingham, Mass.). While maintaining the separation of themagnetic latex with the magnet, the liquid was carefully removed using a10 ml sterile pipette. This washing process was repeated an additionalthree times. After the final wash, the latex was resuspended in 2 ml ofdistilled water. In a separate 50 ml conical tube, 10 mg of avidin-HS(NeutrAvidin, Pierce, Rockford, Ill.) was dissolved in 18 ml of 40 mMTris, 0.15 M sodium chloride, pH 7.5 (TBS). While vortexing, the 2 ml ofwashed magnetic latex was added to the diluted avidin-HS and the mixturemixed an additional 30 seconds. This mixture was incubated at 45° C. for2 hr, shaking every 30 minutes. The avidin magnetic latex was separatedfrom the solution using a magnet and washed three times with 20 ml BBSas described above. After the final wash, the latex was resuspended in10 ml BBS and stored at 4° C.

Immediately prior to use, the avidin magnetic latex was equilibrated inpanning buffer (40 mM Tris, 150 mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH7.5). The avidin magnetic latex needed for a panning experiment (200μl/sample) was added to a sterile 15 ml centrifuge tube and brought to10 ml with panning buffer. The tube was placed on the magnet for 10 minto separate the latex. The solution was carefully removed with a 10 mlsterile pipette as described above. The magnetic latex was resuspendedin 10 ml of panning buffer to begin the second wash. The magnetic latexwas washed a total of 3 times with panning buffer. After the final wash,the latex was resuspended in panning buffer to the starting volume.

Example 4 Selection of Recombinant Polyclonal Antibodies to Bacillusanthracis Sap Antigen

Binding reagents that specifically bind to B. anthracis SAP wereselected from the phage display libraries created from hyperimmunizedmice as described in Example 3.

Panning

First round antibody phage were prepared as described in Example 3 usingBS45 uracil template. Electroporations of mutagenesis DNA were performedyielding phage samples derived from different immunized mice. To createmore diversity in the recombinant polyclonal library, each phage samplewas panned separately.

Before the first round of functional panning with biotinylated SAPantigen, antibody phage libraries were selected for phage displayingboth heavy and light chains on their surface by panning with7F11-magnetic latex (as described in Examples 21 and 22 of U.S. patentapplication Ser. No. 08/835,159, filed Apr. 4, 1997). Functional panningof these enriched libraries was performed in principle as described inExample 16 of U.S. patent application Ser. No. 08/835,159. Specifically,10 μL of 1×10⁻⁶ M biotinylated SAP antigen was added to the phagesamples (approximately 1×10⁻⁸ M SAP final concentration), and themixture allowed to come to equilibrium overnight at 2-8° C.

After reaching equilibrium, samples were panned with avidin magneticlatex to capture antibody phage bound to SAP. Equilibrated avidinmagnetic latex (Example 3), 200 μL latex per sample, was incubated withthe phage for 10 min at room temperature. After 10 min, approximately 9ml of panning buffer was added to each phage sample, and the magneticlatex separated from the solution using a magnet. After a ten minuteseparation, unbound phage was carefully removed using a 10 ml sterilepipette. The magnetic latex was then resuspended in 10 ml of panningbuffer to begin the second wash. The latex was washed a total of threetimes as described above. For each wash, the tubes were in contact withthe magnet for 10 min to separate unbound phage from the magnetic latex.After the third wash, the magnetic latex was resuspended in 1 ml ofpanning buffer and transferred to a 1.5 mL tube. The entire volume ofmagnetic latex for each sample was then collected and resuspended in 200ul 2×YT and plated on 150 mm LB plates as described in Example 3 toamplify bound phage. Plates were incubated at 37° C. for 4 hr, thenovernight at 20° C.

The 150 mm plates used to amplify bound phage were used to generate thenext round of antibody phage. After the overnight incubation, secondround antibody phage were eluted from the 150 mm plates by pipetting 10mL of 2×YT media onto the lawn and gently shaking the plate at roomtemperature for 20 min. The phage samples were then transferred to 15 mldisposable sterile centrifuge tubes with a plug seal cap, and the debrisfrom the LB plate pelleted by centrifuging the tubes for 15 min at 3500rpm. The supernatant containing the second round antibody phage was thentransferred to a new tube.

A second round of functional panning was set up by diluting 100 μL ofeach phage stock into 900 μL of panning buffer in 15 ml disposablesterile centrifuge tubes. Biotinylated SAP antigen was then added toeach sample as described for the first round of panning, and the phagesamples incubated for 1 hr at room temperature. The phage samples werethen panned with avidin magnetic latex as described above. The progressof panning was monitored at this point by plating aliquots of each latexsample on 100 mm LB agar plates to determine the percentage of kappapositives. The majority of latex from each panning (99%) was plated on150 mm LB agar plates to amplify the phage bound to the latex. The 100mm LB agar plates were incubated at 37° C. for 6-7 hr, after which theplates were transferred to room temperature and nitrocellulose filters(pore size 0.45 mm, BA85 Protran, Schleicher and Schuell, Keene, N.H.)were overlaid onto the plaques.

Plates with nitrocellulose filters were incubated overnight at roomtemperature and then developed with a goat anti-mouse kappa alkalinephosphatase conjugate to determine the percentage of kappa positives asdescribed below. Phage samples with lower percentages (<70%) of kappapositives in the population were subjected to a round of panning with7F11-magnetic latex before performing a third functional round ofpanning overnight at 2-8° C. using biotinylated SAP antigen atapproximately 2×10⁻⁹ M. This round of panning was also monitored forkappa positives. Individual phage samples that had kappa positivepercentages greater than 80% were pooled and subjected to a final roundof panning overnight at 2-8° C. at 5×10⁻⁹ M SAP. Antibody genescontained within the eluted phage from this fourth round of functionalpanning were subcloned into the expression vector, pBRncoH3.

The subcloning process was done generally as described in Example 18 ofU.S. patent application Ser. No. 08/835,159. After subcloning, theexpression vector was electroporated into DH10B cells and the mixturegrown overnight in 2×YT containing 1% glycerol and 10 μg/mltetracycline. After a second round of growth and selection intetracycline, aliquots of cells were frozen at −80° C. as the source forSAP polyclonal antibody production. Two polyclonal antibodies,designated IIT004.1 and IIT005.1, were selected from two librariesderived from different sets of spleens. Monoclonal antibodies wereselected from these polyclonal mixtures by plating a sample of themixture on LB agar plates containing 10 μg/ml tetracycline and screeningfor antibodies that recognized SAP.

Detection of Alkaline Phosphatase Conjugates

After overnight incubation of nitrocellulose filters on LB agar plates,filters were carefully removed from the plates with membrane forceps andincubated for 2 hr in 10 mM TRIS, 150 mM NaCl, 10 mM MgCl₂, 0.1 mMZnCl₂, 0.1% polyvinyl alcohol, 1% bovine serum albumin, 0.1% sodiumazide, pH 8.0 (Block buffer). After 2 hr, the filters were incubatedwith goat anti-mouse kappa-AP (Southern Biotechnology Associates, Inc,Birmingham, Ala.) for 2-4 hours. The goat anti-mouse kappa-AP wasdiluted into Block buffer at a final concentration of 1 μg/ml. Filterswere washed three times with 40 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH7.5 (TBST) for 5 min each. After the final wash, the filters weredeveloped in a solution containing 0.2 M 2-amino-2-methyl-1-propanol(JBL Scientific, San Luis Obispo, Calif.), 0.5 M Tris, 0.33 mg/ml nitroblue tetrazolium ((NBT) Fisher Scientific, Pittsburgh, Pa.) and 0.166mg/ml 5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt.

Expression and Purification of Recombinant Antibodies Against SAP

A shake flask inoculum was generated overnight from a −70° C. cell bankin an Innova 4330 incubator shaker (New Brunswick Scientific, Edison,N.J.) set at 37° C., 300 rpm. The inoculum was used to seed a 20 Lfermentor (Applikon, Foster City, Calif.) containing defined culturemedium (Pack et al. (1993) Bio/Technology 11: 1271-1277) supplementedwith 3 g/L L-leucine, 3 g/L L-isoleucine, 12 g/L casein digest (Difco,Detroit, Mich.), 12.5 g/L glycerol and 10 μg/ml tetracycline. Thetemperature, pH and dissolved oxygen in the fermentor were controlled at26° C., 6.0-6.8 and 25% saturation, respectively. Foam was controlled byaddition of polypropylene glycol (Dow, Midland, Mich.). Glycerol wasadded to the fermentor in a fed-batch mode. Fab expression was inducedby addition of L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L duringthe late logarithmic growth phase. Cell density was measured by opticaldensity at 600 nm in an UV-1201 spectrophotometer (Shimadzu, Columbia,Md.). Following run termination and adjustment of pH to 6.0, the culturewas passed twice through an M-210B-EH Microfluidizer (Microfluidics,Newton, Mass.) at 17,000 psi. The high pressure homogenization of thecells released the Fab into the culture supernatant.

The first step in purification was expanded bed immobilized metalaffinity chromatography (EB-IMAC). Streamline™ chelating resin(Pharmacia, Piscataway, N.J.) was charged with 0.1 M NiCl₂ and was thenexpanded and equilibrated in 50 mM acetate, 200 mM NaCl, 10 mMimidazole, 0.01% NaN₃, pH 6.0 buffer flowing in the upward direction. Astock solution was used to bring the culture homogenate to 10 mMimidazole, following which it was diluted two-fold or higher inequilibration buffer to reduce the wet solids content to less than 5% byweight. It was then loaded onto the Streamline column flowing in theupward direction at a superficial velocity of 300 cm/hr. The cell debrispassed through unhindered, but the Fab was captured by means of the highaffinity interaction between nickel and the hexahistidine tag on the Fabheavy chain. After washing, the expanded bed was converted to a packedbed and the Fab was eluted with 20 mM borate, 150 mM NaCl, 200 mMimidazole, 0.01% NaN₃, pH 8.0 buffer flowing in the downward direction.

The second step in the purification used ion-exchange chromatography(IEC). Q Sepharose FastFlow resin (Pharmacia, Piscataway, N.J.) wasequilibrated in 20 mM borate, 37.5 mM NaCl, 0.01% NaN₃, pH 8.0. The Fabelution pool from the EB-IMAC step was diluted four-fold in 20 mMborate, 0.01% NaN₃, pH 8.0 and loaded onto the IEC column. Afterwashing, the Fab was eluted with a 37.5-200 mM NaCl salt gradient. Theelution fractions were evaluated for purity using an Xcell II™ SDS-PAGEsystem (Novex, San Diego, Calif.) prior to pooling. Finally, the Fabpool was concentrated and diafiltered into 20 mM borate, 150 mM NaCl,0.01% NaN₃, pH 8.0 buffer for storage. This was achieved in a SartoconSlice™ system fitted with a 10,000 MWCO cassette (Sartorius, Bohemia,N.Y.). The final purification yields were typically 50%. Theconcentration of the purified Fab was measured by UV absorbance at 280nm, assuming an absorbance of 1.6 for a 1 mg/ml solution.

Culture of Bacillus SPP. and Preparation of Cleared Culture SupernatantAntigen

Nonencapsulated Bacillus anthracis, Sterne strain was obtained from theColorado Serum Company. B. cereus OH599 was the kind gift of Dr. A.Kotiranta, B. globigii was obtained from Dr. L. Larson, and B.thuringiensis 10792 was obtained from the American Type CultureCollection (Manassas, Va.). Organisms were cultured on tryptic soy agarcontaining 5% sheep blood (Hardy Diagnostics, Santa Maria, Calif.) or inbrain heart infusion broth (Becton Dickinson and Company, Cockeysville,Md.) at 37° C.

For preparation of cleared culture supernatant antigens, B. anthracis,Sterne strain was grown in brain heart infusion broth at 37° C. withaeration for 24 h. A sample of the culture was serially diluted 10-foldin sterile 0.01M phosphate buffered saline, pH 7.4 (PBS) and 100 μl ofeach dilution was plated on a blood agar plate for determination of thenumber of viable organisms. The culture was subjected to centrifugationat 10,000×g for 20 min at 4° C. using a J2-21 centrifuge (Beckman,Fullerton, Calif.). The supernatant was transferred to a sterile bottleand a protease inhibitor cocktail (Sigma-Aldrich, Inc) was added. Thesample was filtered using a 0.2 μm pore-size membrane filter unit(Millipore Corp., Bedford, Mass.) then dialyzed in 4 L of 0.01M PBS, pH7.4, 2 mM EDTA, 0.1 mM phenymethylsulfonyl fluoride at 4° C. with fourbuffer changes in 24 h. The dialyzed sample was aliquoted and stored at−80° C. The concentration of SAP in the cleared culture supernatant wasquantified by a sandwich enzyme-linked immunosorbant assay usingpurified recombinant SAP as a standard. Analysis of the amount of SAPrecovered from the culture supernatant indicated that 1 ng of SAPcorresponded to approximately 2.9×10³ organisms (i.e. 0.35 pg/organism).

Example 5 Selection of Monoclonal Antibodies to SAP from the RecombinantPolyclonal Antibody Mixtures

Monoclonal antibodies against SAP were isolated from clones containingthe recombinant polyclonal mixtures (Example 4) by plating a dilutedsample of the mixture on LB agar plates containing 10 μg/mltetracycline. Individual colonies were then tested for the ability toproduce antibody that recognized recombinant SAP using surface plasmonresonance (BIACORE) (BIACORE, Uppsala, Sweden). Small scale productionof these monoclonal antibodies was accomplished using a Ni-chelatebatch-binding method (see below). Antibodies isolated from this methodwere diluted 1:3 in HBS-EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mMEDTA, 0.005% polysorbate 20 (v/v)), captured with a goat anti-mousekappa antibody (Southern Biotechnology Associates, Inc, Birmingham,Ala.) coupled to a BIACORE CM5 sensor chip, and tested for the abilityto bind recombinant SAP. Antibodies that bound SAP were then evaluatedusing BIACORE epitope mapping analysis. Antibodies that bound distinctepitopes were produced on a larger scale and then conjugated to biotinand alkaline phosphatase. These conjugates were then tested in an ELISAassay to determine the sensitivity and utility of these antibodies.

Minipreparation of Monoclonal Antibodies by Ni-Chelate Batch-BindingMethod

Individual colonies were isolated from the recombinant polyclonalmixtures (Example 4) and used to inoculate 3 ml cultures of 2×YT mediumcontaining 1% glycerol supplemented with 10 μg/ml tetracycline. Thesecultures were grown in an Innova 4330 incubator shaker (New BrunswickScientific, Edison, N.J.) set at 37° C., 300 rpm. The next morning 0.5ml of each culture was used to inoculate shake flasks containing 50 mlof defined medium, (Pack et al. (1993) Bio/Technology 11: 1271-1277)supplemented with 3 g/L L-leucine, 3 g/L L-isoleucine, 12 g/L caseindigest (Difco, Detroit, Mich.), 12.5 g/L glycerol and 10 μg/mltetracycline. These cultures were shaken at 300 rpm, 37° C. until anoptical density of 4 was reached at 600 nm. Fab expression was theninduced by adding L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 g/L andshifting the temperature to 23° C. with overnight shaking. The next daythe following was added to the 50 ml cultures: 0.55 ml of 1 M imidazole,5 ml B-PER (Pierce, Rockford, Ill.) and 2 ml Ni-chelating resin(Chelating Sepharose FastFlow™ resin Pharmacia, Piscataway, N.J.). Themixture was shaken at 300 rpm, 23° C. for 1 hour after which timeshaking was stopped and the resin allowed to settle to the bottom of theflasks for 15 minutes.

The supernatant was then poured off and the resin resuspended in 40 mlof BBS (20 mM borate, 150 mM NaCl, 0.1% NaN₃, pH 8.0) containing 10 mMimidazole. This suspension was transferred to a 50 ml conical tube andthe resin washed a total of 3 times with BBS containing 10 mM imidazole.Washing was accomplished by low speed centrifugation (1100 rpm for 1minute), removal of supernatant and, resuspension of the resin in BBScontaining 10 mM imidazole. After the supernatant of the final wash waspoured off, 0.5 ml of 1 M imidazole was added to each tube, vortexbriefly, and transferred to a sterile microcentrifuge tube. The sampleswere then centrifuged at 14 krpm for 1 minutes and the supernatanttransferred to a new microcentrifuge tube. Antibodies contained in thesupernatant were then analyzed for binding to SAP using a BIACORE(BIACORE, Uppsala, Sweden).

Selection and Cloning of a Recombinant Polyclonal Antibody Complementaryto IIT005.1.13 and IIT005.1.C.11 Monoclonal Antibodies

A monoclonal antibody designated IIT004.1.12 was selected from thepolyclonal library designated IIT004.1, biotinylated, and 15 μl of a10⁻⁶ M solution was mixed with soluble recombinant SAP antigen (7.5 μlof a 10⁻⁷ M solution). This mixture was incubated for 15 minutes at roomtemperature. Fifteen microliters of each mixture was added to 50 μl ofphage library IIT005.1 diluted in 1 ml panning buffer (40 mM Tris, 150mM NaCl, 20 mg/ml BSA, 0.1% Tween 20, pH 7.5) and incubated overnight at2-8° C. Final concentrations were 10⁻⁸ M biotinylated monoconal antibodyand 5×10⁻¹⁰ M SAP. The sample was panned with avidin magnetic latex andplated as described in Example 4. The eluted phage were subjected toanother round of selection using these conditions and the resultingpolyclonal library was designated IIT005.1.C. Two monoclonal antibodiesdesignated IIT005.1.13 and IIT005.1.C.11 were selected from the IIT005.1and IIT005.1.C libraries respectively, biotinylated and complexed withSAP using the conditions described above. Complementary polyclonalantibodies were selected as described above from phage library IIT005.1using monoclonal antibodies IIT005.1.13 and IIT005.1.C.11. Thesecomplementary polyclonal antibodies were designated IIT005.1.13.1 andIIT005.1.C.11.1 and were subcloned as described in Example 18 of U.S.patent application Ser. No. 08/835,159.

Example 6 Specificity of Monoclonal/Polyclonal Antibodies to SapDetermined by Western Blot and Indirect Immunofluorescence Analysis

The specificity of monoclonal and polyclonal antibodies against B.anthracis, Sterne strain SAP was visualized by Western blot analysis.Recombinant antibodies against SAP were tested for reactivity torecombinant SAP as well as to SAP isolated from the cleared culturesupernatant of Bacillus anthracis Sterne strain. Cross reactivity toother Bacillus strains was also tested. Culture supernatant proteins andwhole cell lysates of B. anthracis, Sterne strain, B. cereus OH599, B.globigii and B. thuringiensis 10792 equivalent to 108 organisms wereseparated by electrophoresis in 4-20% TRIS-glycine SDS-polyacrylamidegels (Novex, San Diego, Calif.) under reducing conditions. The proteinswere transferred to ProBlott™ membranes (Applied Biosystems, FosterCity, Calif.) using 10 mM CAPS/10% methanol transfer buffer. Themembranes were blocked in 10 mM TRIS, 150 mM NaCl, 10 mM MgCl₂, 0.1 mMZnCl₂, 0.1% polyvinyl alcohol, 1% bovine serum albumin, 0.1% sodiumazide, pH 8.0 (Block buffer) for 1 h at room temperature.

The membranes were then incubated in 5 μg/ml of monoclonal orrecombinant polyclonal antibody diluted in Block buffer for 1 h and thenwashed three times with 40 mM TRIS, 150 mM NaCl, 0.05% Tween 20, pH 7.5(TBST) (Fisher Chemical, Pittsburgh, Pa.) for 5 min each. After washing,the membranes were incubated in rabbit anti-mouse IgG (H&L)-alkalinephosphatase conjugate (Southern Biotechnology, Inc, Birmingham, Ala.)diluted 1:1000 in Block buffer. The membranes were washed three timeswith TBST for 5 min each and developed in a solution containing 0.2 M2-amino-2-methyl-1-propanol (JBL Scientific, San Luis Obispo, Calif.),0.5 M TRIS, 0.33 mg/ml nitro blue tetrazolium ((NBT) Fisher Scientific,Pittsburgh, Pa.) and 0.166 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate,p-toluidine salt.

The anti-SAP recombinant polyclonal antibodies reacted with recombinantSAP, SAP protein isolated from the culture supernatant, and the cellpellet of B. anthracis, Sterne strain. The antibodies did not react withany proteins in the culture supernatant or cell pellet of the otherBacillus species tested (B. cereus and thuringiensis). A goatanti-anthrax polyclonal serum was used to demonstrate cross-reactivityof B. anthracis antibodies with proteins of other Bacillus species (datanot shown). Conjugates alone served as negative controls.

The specificity of antibodies against B. anthracis, Sterne strain wasalso tested by indirect immunofluorescence. Localization of SAP to theouter membrane of unencapsulated B. anthracis, Sterne strain wasdemonstrated using an indirect immunofluorescence technique. B.anthracis, B. cereus, and B. thuringiensis were washed and resuspendedin PBS to yield 1×10⁸ organisms per ml. Four microliters of thesuspensions were applied to wells of an eight well microscope slide andallowed to air dry. The slides were lightly heated to fix the smears tothe slide and covered with 0.1 mg/ml of antibody diluted in PBScontaining 1% BSA. The smears were incubated with antibody for 1 h at37° C. in a moist chamber. After washing the slides three times bysoaking in PBS for 5 min each, the smears were covered with fluoresceinisothiocyanate-conjugated rabbit anti-mouse IgG (H&L) F(ab′)₂ (ZymedLaboratories, Inc., South San Francisco, Calif.) diluted 1:80 in PBS, 1%BSA, 0.05% Evans Blue (Sigma). The slides were incubated for 1 h at 37 Cin a moist chamber then washed as described above. After a final wash indeionized water, the slides were allowed to air dry in the dark.Coverslips were mounted using a 90% glycerol mounting medium containing10 mg/ml p-phenylenediamine, pH 8.0.

The slides were examined for fluorescent organisms using anepifluorescence microscope with a 63× objective lens (Leitz WetzlerGermany). The recombinant polyclonal antibody (ITT005.1) demonstrated 4+fluorescence with unencapsulated B. anthracis and did not react with B.cereus, or B. thuringiensis. Negative controls includedfluorescein-conjugated antibody alone, and a murine polyclonal antiserumspecific for B. anthracis, Sterne strain spore coat proteins.

Example 7 Sensitivity and Specificity of an ELISA Plate Assay forDetection of B. anthracis SAP

This Example demonstrates that an ELISA assay using the reagents andmethods of the invention are not only highly sensitive for B. anthracis,but are also highly specific for this particular Bacillus species.

The sensitivity and specificity of various monoclonal/recombinantpolyclonal antibody pairs were determined by performing a sandwich assayusing biotinylated monoclonal antibodies and alkalinephosphatase-conjugated recombinant polyclonal antibodies. Assays wereperformed with NeutraAvidin or streptavidin coated plates, such asReacti-Bind™ streptavidin coated polystyrene 96 well plates (PierceChemical, Rockford, Ill.). After washing the 96 well plate with BBS (20mM borate, 150 mM NaCl, 0.01% NaN₃, pH 8.0) containing 0.02% TWEEN-20,biotinylated monoclonal antibodies (50 μL of 2.5 μg/mL diluted in Blockbuffer (10 mM Tris, 150 mM NaCl, 10 mM MgCl₂, 0.1 mM ZnCl₂, 0.1%polyvinyl alcohol, 1% bovine serum albumin, 0.1% sodium azide, pH 8.0))were added to the wells. The plate was incubated at room temperature for1 hr.

The plate was then washed, after which various dilutions (10 ng/ml to0.625 ng/ml) of soluble SAP antigen (50 μL of recombinant SAP or SAP inculture supernatants (as prepared in Example 4) were added in duplicateto the biotinylated monoclonal wells. The plates were incubated for onehour at room temperature or overnight at 2-8° C., after which the platewas washed. The appropriate recombinant polyclonal antibody-alkalinephosphatase conjugate (50 μL of 2.5 μg/mL diluted in Block) was addedand incubated at room temperature for 1 hr. After 1 hr, the plate waswashed and developed using the ELISA Amplification System (Gibco BRL,Gaithersburg, Md.) according to the manufacturer's instructions.

Results from several assays are compiled in accompanying tables 2-5.These data indicate that the assays can detect less than 0.625 ng of SAPprotein. This amount of SAP corresponds to approximately 1.8×10³Bacillus anthracis organisms per ml. Significantly, little or no crossreactivity to other related Bacillus species was detected.

TABLE 2 IIT005.1.C.11-BIOTIN WITH IIT005.1.C11.1-AP B. anthracisUndiluted culture SAP Bacillus (cfu/ml) A490 (ng/mL) A490 species A49028330 3.4 10 3.55 cereus 0.28 14165 2.8 5 3.45 thuringiensis 0.27 70832.14 2.5 2.94 subtilis niger 0.55 3541 1.56 1.25 2.01 subtilis 0.51 17701.17 0.625 1.51 BHI broth 0.48 0 0.92 0 0.92 media

TABLE 3 IIT005.1.13-BIOTIN WITH IIT005.1.13.1-AP B. anthracis Undilutedculture Bacillus (cfu/ml) A490 species A490 28330 3.13 cereus 0.28 141652.21 thuringiensis 0.27 7083 1.5 subtilis niger 0.48 3541 0.99 subtilis0.5 1770 0.78 BHI broth 0.423 0 0.55 media

TABLE 4 IIT005.1.C.11-BIOTIN WITH IIT005.1-AP B. anthracis Undilutedculture SAP Bacillus (cfu/ml) A490 (ng/mL) A490 species A490 28330 2.8710 3.4 cereus 0.09 14165 1.698 5 2.56 thuringiensis 0.14 7083 1 2.5 1.49subtilis niger 0.13 3541 0.55 1.25 0.82 subtilis 0.14 1770 0.35 0.6250.49 BHI broth 0.148 0 0.14 0 0.19 media

TABLE 5 IIT005.1.13-BIOTIN WITH IIT005.1-AP B. anthracis Undilutedculture Bacillus (cfu/ml) A490 species A490 28330 1.77 cereus 0.08514165 0.99 thuringiensis 0.121 7083 0.54 subtilis niger 0.125 3541 0.34subtilis 0.124 1770 0.23 BHI broth 0.125 0 0.14 media

These results demonstrate that four different monoclonal/recombinantpolyclonal antibody preparations exhibit great sensitivity for B.anthracis while not cross reacting with other Bacillus species.

Example 8

Assay for the Detection of Anthrax Infection in Humans

Blood samples from individuals suspected of exposure to B. anthracis areobtained by venous puncture and collected in tubes with (for plasmaseparation) or without (for serum separation) anti-coagulants present.Volumes of sample (100 μl) are contacted separately with either affinityagent for antibody detection or capture reagent for the detection of SAPantigen. These reagents are separately immobilized in different wells ina 96-well microtiter plate. The microtiter wells are coated so that theybind biotinylated molecules (REACTI-BIND™ NEUTRAVIDIN™, Pierce,Rockford, Ill.). Biotinylated SAP antigen and biotinylated captureantibody are added to their respective wells in volumes of 100 μl/wellat concentrations of 2 μg/ml. After one hour of incubation at roomtemperature, the wells are washed with BBS to remove unbound reagent andthe samples are added to each well. Negative control samples notcontaining SAP antigen or antibody to SAP antigen as well as positivecontrol samples containing either SAP antigen or antibody to SAP antigenare added to separate wells prepared as described. The samples andcontrols are incubated with the immobilized reagents for one hour atroom temperature, the samples are removed by aspiration and the wellsare each washed using several one-ml volumes of BBS containing 0.05%TRITON X-100 detergent with aspiration between the addition of eachvolume of wash solution.

For the detection of human antibody in wells containing the biotinylatedSAP antigen, alkaline phosphatase conjugates of mouse monoclonalantibodies specific for either human IgG (clone G18-145) or human IgM(clone G20-127, both from BD Biosciences/Pharmingen, San Diego, Calif.)are added in conjugate diluent at concentrations of 1 μg/ml andincubated for one hour at room temperature. For the detection of SAPantigen in samples one of the monoclonal antibodies described in Example5 is biotinylated and used as the capture reagent and the other isconjugated to alkaline phosphatase and used as the detection reagentusing the same assay steps as described for the detection of humanantibodies. The wells are washed with BBS containing 0.05% TRITON X-100as described. The amount of bound enzyme activity is detected by ELISAamplification reagents (Gibco BRL, Gaithersburg, Md.) according to themanufacturer's instructions. The absorbance at 490 nm is measured usinga microtiter plate reader. From testing a population of clinical samplesfrom healthy people a positive cutoff value is determined so that 95% ofthe normal samples values fall beneath the cutoff value. Any well oraverage of replicate wells that exceeds the cutoff value is considered apositive result. Higher specificities of 98% or 99% can be achieved bydetermining the appropriate cutoff value from the results with normalsamples. The values measured in the positive control wells can be usedto provide a rough calibration of the assay response so that the cutoffvalue can be determined as a percentage of the difference between thenegative and positive control values. The presence of either human IgMor human IgG specific for SAP is determined as well as the presence ofSAP antigen. The presence of any one of these in a human blood sample isindicative of infection with B. anthracis. The presence of either SAPantigen or specific IgM indicates that the patient has recentlydeveloped the infection. The presence of both specific IgM and IgGindicates an active infection that has progressed to a secondary immuneresponse. The presence of specific IgG alone indicates that the patientwas exposed, developed a secondary immune response and may have clearedthe organism.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference for allpurposes.

1-33. (canceled)
 34. A kit for the detection of an anti-B. anthracisantibody in a biological sample, the kit comprising an affinity agentnon-diffusively immobilized on a solid support, the affinity agentcomprising at least 10 contiguous amino acids of SEQ ID NO:1, whereinthe affinity agent forms a first complex with the anti-B. anthracisantibody if the anti-B. anthracis antibody is contacted to the affinityagent.
 35. (canceled)
 36. The kit of claim 34, wherein the affinityagent comprises at least 10 contiguous amino acids of amino acids 180 to700 of SEQ ID NO:1.
 37. The kit of claim 34, wherein the affinity agentcomprises SEQ ID NO:1.
 38. The kit of claim 34, wherein the solidsupport comprises a microtiter plate and the affinity agent is presentin the wells of the microtiter plate.
 39. The kit of claim 34, furthercomprising a first detection reagent.
 40. The kit of claim 39, whereinthe first detection reagent comprises a detection antibody that binds tothe first complex.
 41. The kit of claim 40, wherein the detectionantibody is labeled.
 42. The kit of claim 41, where in the label isselected from the group consisting of enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds,phosphorescent compounds and bioluminescent compounds.
 43. The kit ofclaim 42, wherein the detection antibody specifically binds to a humanantibody.
 44. The kit of claim 43, wherein the human antibodies detectedcomprise IgG and IgM isotypes.
 45. The kit of claim 34, furthercomprising a capture reagent immobilized on a solid support, wherein thecapture reagent forms a second complex with a B. anthracis surface arrayprotein if the surface array protein is present in the sample.
 46. Thekit of claim 45, wherein the capture reagent and affinity agent areimmobilized on the same solid support.
 47. The kit of claim 45, whereinthe capture reagent is immobilized on a microtiter dish.
 48. The kit ofclaim 45, wherein the capture reagent is a third antibody.
 49. The kitof claim 48, wherein the capture reagent is a recombinant polyclonalantibody.
 50. The kit of claim 48, wherein the capture reagent is amonoclonal antibody.
 51. The kit of claim 45, wherein the kit furthercomprises a positive control that comprises a polypeptide that comprisesan antigenic determinant of a B. anthracis surface array protein. 52.The kit of claim 51, herein the antigenic determinant comprises an aminoacid sequence of SEQ ID NO:1.
 53. The kit of claim 52, wherein theantigenic determinant comprises at least 10 contiguous amino acids ofamino acids 180 to 700 of SEQ ID NO:1.
 54. The kit of claim 45, furthercomprising a second detection reagent.
 55. The kit of claim 54, whereinthe second detection reagent comprises a fourth antibody that binds tothe second complex.
 56. The kit of claim 55, wherein the fourth antibodyis labeled.